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1521-0081/68/4/954–1013$25.00 http://dx.doi.org/10.1124/pr.115.011395 PHARMACOLOGICAL REVIEWS Pharmacol Rev 68:954–1013, October 2016 Copyright © 2016 by The Author(s) This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.

ASSOCIATE EDITOR: RICHARD DEQUAN YE -Like -1 and Its Class B G –Coupled Receptors: A Long March to Therapeutic Successes

Chris de Graaf, Dan Donnelly, Denise Wootten, Jesper Lau, Patrick M. Sexton, Laurence J. Miller, Jung-Mo Ahn, Jiayu Liao, Madeleine M. Fletcher, Dehua Yang, Alastair J. H. Brown, Caihong Zhou, Jiejie Deng, and Ming-Wei Wang Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (C.d.G.); School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom (D.D.); Drug Discovery Biology Theme and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia (D.W., P.M.S., M.M.F.); Protein and Peptide Chemistry, Global Research, Novo Nordisk A/S, Måløv, Denmark (J.La.); Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (L.J.M.); Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, Texas (J.-M.A.); Department of Bioengineering, Bourns College of Engineering, University of California at Riverside, Riverside, California (J.Li.); National Center for Drug Screening and CAS Key Laboratory of Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (D.Y., C.Z., J.D., M.-W.W.); Heptares Therapeutics, BioPark, Welwyn Garden City, United Kingdom (A.J.H.B.); and School of Pharmacy, Fudan University, Zhangjiang High-Tech Park, Shanghai, China (M.-W.W.) Downloaded from

Abstract...... 956 I. Introduction...... 956 II. Glucagon-Like Peptide-1...... 957 A. Discovery...... 957

B. Physiology...... 957 by guest on September 27, 2021 1. Effect on ...... 958 2. Effect on Gastric Emptying...... 958 3. Effect on Food Intake...... 959 4. Effect on Cardiovascular Activity...... 959 5. Effect on Immune Response...... 959 6. Effect on Function...... 960 7. Effect on Nervous System...... 960 C. Structure...... 960 1. a-Helical C-Terminal Region...... 962 2. Flexible N-Terminal Region...... 962 3. Interaction between the C-Terminal Helix and ECD...... 963 4. Interaction between Flexible N terminus and 7TMD...... 964 D. Mutagenesis...... 964 1. Truncated GLP-1 Analogs...... 964 2. Chimeric GLP-1 Analogs...... 965 3. Substituted GLP-1 Analogs...... 965 a. GLP-1 hydrophobic region I...... 965 b. GLP-1 hydrophobic region II...... 966 c. Polar residues in a-helix...... 966 d. N-capping motif in GLP-1...... 966

This work was supported by National Health and Family Planning Commission of China [2012ZX09304-011, 2013ZX09401003-005, 2013ZX09507001, and 2013ZX09507-002 to M.-W.W.]; Chinese Academy of Sciences [to M.-W.W.]; Shanghai Science and Technology Development Fund [15DZ2291600 to M.-W.W.]; Thousand Talents Program in China [to M.-W.W.]; the CAS-Novo Nordisk Research Fund [to D.Y.]; National Natural Science Foundation of China [81373463 to D.Y.]; National Health and Medical Research Council of Australia (NHMRC) Principal Research Fellowship [to P.M.S.]; NHMRC Program [Grant 1055134 to P.M.S.]; NHMRC Career Development Fellowship [to D.W.]; NHMRC Project [Grant 1061044 to P.M.S. and Grant 1065410 to D.W.]; Welch Foundation [AT-1595 to J.-M.A.]; and Juvenile Diabetes Research Foundation [37-2011-20 to J.-M.A.]. C.d.G., D.D., and D.W. contributed equally to this work. Address correspondence to: Dr. Ming-Wei Wang, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Shanghai 201203, China. E-mail: [email protected] dx.doi.org/10.1124/pr.115.011395.

954 Glucagon-Like Peptide-1 and Its Receptors 955

e. N terminus of GLP-1...... 966 III. Glucagon-Like Peptide 1 Receptor ...... 967 A. Discovery...... 967 1. Receptor Cloning...... 967 2. Receptor Expression...... 967 3. Receptor Biosynthesis...... 967 B. Structure...... 968 1. N-Terminal Domain...... 969 a. Structure determination of the N-terminal ECD...... 969 b. Site-directed mutagenesis of the ECD...... 969 2. Seven-Transmembrane Domain...... 969 a. Structure of the 7TMD...... 969 b. Mutagenesis of the 7TMD...... 976 i. TM1 (S1291.24b-L1671.62b)...... 976 ii. ICL1 (G168-C174)...... 976 iii. TM2 (T1752.45b-L2012.71b)...... 976 iv. ECL1 (K202-L218)...... 977 v. TM3 (S2193.22b-A2563.59b)...... 977 vi. ICL2 (F257-I265)...... 978 vii. TM4 (F2664.42b-Y2914.62b)...... 978 viii. ECL2 (E292-Y305)...... 978 ix. TM5 (W3065.36b-K3365.66b)...... 978 x. ICL3 (A337-T343)...... 979 xi. TM6 (D3446.33b-F3696.58b)...... 979 xii. ECL3 (V370-G377)...... 979 xiii. TM7 (Y3787.33b-Y4027.57b)...... 979 xiv. C terminus (C404-S463)...... 979 C. Receptor Function ...... 979 1. Signaling...... 980 a. Recombinant cells...... 980 b. Pancreatic b cells...... 980 i. ...... 980 ii. Insulin Synthesis and Storage...... 981 iii. b Cell Survival, Proliferation, and Neogenesis...... 981 c. Extrapancreatic signaling...... 982 i. ...... 982 ii. Kidney...... 982 iii. Adipocytes...... 983 iv. Nervous System...... 983 v. Cardiovascular System...... 983 2. -Directed Signal Bias...... 983 a. Peptide-mediated signal bias...... 983 b. Nonpeptide-mediated bias...... 984

ABBREVIATIONS: Ab, amyloid b protein; AD, Alzheimer’s disease; Aib, aminoisobutyric acid; AMPK, AMP-activated protein kinase; BCM, b cell mass; BETP, 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine; CD, circular dichroism; CHI, congenital hyperinsulinism; CHL, Chinese hamster lung; CHO, Chinese hamster ovary; CICR, -induced calcium release; CNS, central nervous system; CREB, cAMP response element-binding protein; CRF1R, corticotropin-releasing factor-1 receptor; DPC, dodecylphosphocholine; DPP-4, dipeptidyl peptidase-4; ECD, extracellular domain; ECL, extracellular loop; Emax,maximalresponse;Epac,exchangeprotein activated by cAMP; ER, ; ERK, extracellular regulated kinase;FDA,FoodandDrugAdministration; GCGR, ; GIP, gastric inhibitory polypeptide; GIPR, GIP receptor; GLP, glucagon-like peptide; GLP-1R, GLP-1 receptor; GPCR, –coupled receptor; GRK, G protein– coupled receptor kinase; HbA1c, hemoglobin A1c; HEK, human embryonic kidney; hGLP-1R, human GLP-1R; iCa2+, intracellular calcium; ICL, intracellular loop; IP1, intervening peptide 1; IRS2, substrate 2; KATP, ATP-sensitive potassium; MAPK, -activated protein kinase; NEP 24.11, neutral endopeptidase 24.11; NHE3, Na+/H+ exchanger isoform 3; PACAP, pituitary adenylate cyclase–activating polypeptide; PD, Parkinson’sdisease;PDB,ProteinDataBank;PDX-1,pancreaticandduodenal homeobox-1; PET, positronemissiontomography;PI3K, phosphatidylinositol 3-kinase; PKA, ; PKB, protein kinase B; PKC, protein kinase C; PMA, phorbol myristate acetate; PPAR, peroxisome proliferator-activated receptor; Rim, Rab-3–interacting molecule; SAR, structure–activity relationship; SNP, single-nucleotide polymorphism; SPECT, single-photon emission-computed tomography; SRP, signal recognition particle; SUR1, receptor; T2DM, mellitus; TM, transmembrane; 7TM, seven transmembrane; 7TMD, 7TM domain. 956 de Graaf et al.

3. Molecular Basis for Signal Bias...... 984 4. Allosteric Modulation...... 985 D. Receptor Regulation ...... 988 1. Receptor Desensitization...... 988 2. Underlying Mechanisms...... 988 a. GLP-1R phosphorylation...... 988 b. GRKs...... 989 c. Second messenger protein kinases...... 989 3. In Vivo Evidence of Desensitization...... 989 4. Receptor Internalization...... 990 5. Receptor Resensitization...... 991 IV. Pharmacological Tools ...... 991 A. Traditional Bioassays...... 991 B. Other Bioassays ...... 991 1. Receptor Trafficking...... 991 2. Immunoassays...... 991 3. Pancreatic b Cell Regeneration...... 992 C. Molecular Imaging...... 992 V. Pharmaceutical Development and Therapeutics ...... 994 A. Peptidic Analogs...... 994 1. GLP-1 Analogs...... 994 2. Experience...... 995 3. GLP-1 Mimetics...... 996 B. Nonpeptidic Modulators ...... 996 C. Currently Approved Clinical Applications ...... 998 1. Selection...... 999 2. Efficacy...... 999 3. Benefits...... 1000 D. Potential Applications for Other Diseases ...... 1000 1. Neurologic Disorders...... 1000 2. Oncological Association...... 1001 E. GLP-1R Antagonists ...... 1002 VI. Conclusions ...... 1004 Acknowledgments ...... 1004 References ...... 1004

Abstract——Theglucagon-likepeptide(GLP)-1receptor structures of the 7-helical transmembrane domain (GLP-1R) is a class B G protein–coupled receptor (GPCR) of class B GPCRs, have provided the basis for a two- that mediates the action of GLP-1, a peptide domain–binding mechanism of GLP-1 with its cognate secreted from three major tissues in humans, enteroendocrine receptor. Although efforts in discovering therapeutically L cells in the distal intestine, a cells in the , and viable nonpeptidic GLP-1R have been hampered, the central nervous system, which exerts important small-molecule modulators offer complementary chemical actions useful in the management of type 2 diabetes tools to peptide analogs to investigate ligand-directed mellitus and obesity, including glucose homeostasis and biased cellular signaling of GLP-1R. The integrated regulation of gastric motility and food intake. Peptidic pharmacological and structural information of different analogs of GLP-1 have been successfully developed with GLP-1 analogs and homologous receptors give new enhanced and pharmacological activity. insights into the molecular determinants of GLP-1R Physiologic and biochemical studies with truncated, ligand selectivity and functional activity, thereby chimeric, and mutated and GLP-1R variants, providing novel opportunities in the design and together with ligand-bound crystal structures of the development of more efficacious agents to treat metabolic extracellular domain and the first three-dimensional disorders.

I. Introduction which has multiple therapeutic effects useful in the man- Glucagon-like peptide (GLP)-1 is a gastrointestinal agement of type 2 diabetes mellitus (T2DM). These in- secreted from three major tissues in clude most prominently a glucose-dependent insulinotropic humans, enteroendocrine L cells in the distal intestine, function and other actions on glucose homeostasis, as well a cells in the pancreas, and the central nervous system, as benefits to gastric emptying and appetite regulation Glucagon-Like Peptide-1 and Its Receptors 957 valuable in reducing food intake and body weight. This GLP-1 and GLP-2 were confirmed from cloned hamster hormone exerts its effects by binding to and activating a and human preproglucagon cDNAs, but only GLP-1 was class B G protein–coupled receptor (GPCR), namely, GLP-1 able to stimulate insulin secretion (Bell et al., 1983a, receptor (GLP-1R). We review the current understanding 1983b; Mojsov et al., 1987). The is of the structures of GLP-1 and GLP-1R, the molecular basis expressed in the a cells of the pancreas, the L cells of the of their interaction, and the signaling events associated intestine, and in the caudal brainstem and with it. We also discuss the peptide analogs and non- (Mojsov et al., 1986; Drucker and Asa, peptidic ligands that have been developed to target GLP- 1988) (Fig. 1). Although its transcription produces the 1R, the molecular basis of their action, and the implications same single mRNA in these cell types, the 180-residue for ligand-biased activity and allosteric regulation of this preproglucagon protein translated from it is cleaved hormone-receptor system. Some of these GLP-1R agonists differently in the pancreas than in the intestine (and are already in clinical use, with many more currently being brain) by differential posttranslational processing: the developed, and likely to provide enhancements in their former releases glicentin-related pancreatic peptide, ease of administration, tolerability, and effectiveness. glucagon, intervening peptide 1 (IP1), and major pro- glucagon fragment (containing GLP-1, IP1, and GLP-2 II. Glucagon-Like Peptide-1 as a single fusion peptide), whereas the latter releases glicentin, , GLP-1, IP1, and GLP-2 (Mojsov A. Discovery et al., 1986) (Fig. 1). Endogenous GLP-1 exists in two GLP-1 is a member of the family of gastroin- forms: one corresponds to proglucagon 78–107 with its testinal (Creutzfeldt, 1979; Baggio and Drucker, C-terminal Arg amidated, that is, GLP-17–36 amide,andthe 2007; Campbell and Drucker, 2013; Heppner and Perez- other is longer and not amidated, GLP-17–37 (Holst et al., Tilve, 2015). In 1906, Moore and his colleagues tested 1987; Orskov et al., 1989). Both have similar biologic the hypothesis that the pancreas might be stimulated activities, although the amide form may have slightly by factors from the gut to help disposal of nutrients and improved stability in the circulation (Wettergren et al., started using porcine small intestine extract to treat 1998). GLP-1, but not GLP-2, was later demonstrated to diabetic patients (Moore, 1906). In 1928, Zunc and enhance glucose-stimulated insulin secretion in response LaBarre were able to show a hypoglycemic effect follow- to nutrient ingestion (Schmidt et al., 1985; Kreymann ing injection of extracted from the small intes- et al., 1987; Mojsov et al., 1987; Orskov et al., 1987). tinal mucosa, and this effect was mediated through the The half-life of GLP-1 peptide in the circulation is pancreas (Zunz and LaBarre, 1928). Subsequently, the very short (less than 2 minutes). Its rapid inactivation term incrétine (incretin) was introduced by LaBarre for a is mainly due to the cleavage of two amino acids from substance extracted from the upper gut mucosa, which the N terminus by the ubiquitous proteolytic enzyme produces , but does not stimulate pancre- dipeptidyl peptidase-4 (DPP-4) (Deacon et al., 1995b) atic exocrine secretion (LaBarre, 1932). It was later (Fig. 1). In addition, a membrane-bound zinc metal- observed that orally administered glucose evoked a much lopeptidase, neutral endopeptidase 24.11 (NEP 24.11), stronger insulin release than that induced by i.v. injected has also been shown to cleave GLP-1 at its C terminus glucose, supporting the concept of an entero-insular axis, both in vitro and in vivo (Plamboeck et al., 2005). The that is, gut factor–stimulated insulin secretion (Elrick metabolites of GLP-1 are then subject to renal clearance et al., 1964; McIntyre et al., 1964; Perley and Kipnis, by several mechanisms (Ruiz-Grande et al., 1993). 1967). The first discovered incretin hormone was gastric inhibitory polypeptide (GIP), which was isolated from B. Physiology crude extracts of the porcine small intestine for its ac- The main action of GLP-1 is to work as an incretin, tivity to inhibit gastric acid secretion (Brown et al., 1975). that is, as a gut-derived hormone capable of potentiat- This was followed by the observation that GIP could also ing insulin secretion in the presence of high plasma stimulate insulin secretion in animals and humans, and glucose levels. The incretin concept came from observa- thus, it was later renamed as glucose-dependent insuli- tions that insulin release was higher when the glucose notropic polypeptide, while retaining the same acronym was administered orally rather than i.v., even when the (Dupre et al., 1973; Elahi et al., 1979; Sarson et al., 1984; same plasma glucose concentration was achieved from Creutzfeldt and Ebert, 1985). GIP is released from the K both routes (McIntyre et al., 1964; Perley and Kipnis, cells of the small intestine. However, antibodies raised 1967). Although many hormones were originally sus- against GIP did not abolish the incretin effect, implying pected to contribute to the incretin effect, the current the existence of other prominent gut insulinotropic view is that GLP-1 and GIP are responsible for most factors (Ebert and Creutzfeldt, 1982). incretin activity normally observed (Holst and Orskov, In 1981, GLP-1, the second incretin hormone, was 2001; Vilsboll and Holst, 2004; Creutzfeldt, 2005; identified in the translational products of mRNAs Campbell and Drucker, 2013). Oral administration of isolated from the pancreatic islets of anglerfish (Lund glucose results in a two- to threefold greater insulin et al., 1981; Shields et al., 1981). Subsequently, both secretion than i.v. glucose. Both GLP-1 and GIP can 958 de Graaf et al.

Fig. 1. Gene structure, expression, processing, degradation, and elimination of proglucagon. The proglucagon gene is located in human 2 and transcribed as one single mRNA in three major tissues, namely, the pancreas, the intestine, and the CNS. The mRNA is first translated into one single protein and then processed by prohormone convertase (PC) in different tissues. In the pancreatic a cells, proglucagon protein is processed by PC2 into glicentin-related polypeptide (GRPP), glucagon (Gluc), intervening peptide-1 (IP-1), and major proglucagon fragment, whereas in L cells of the small intestine and the brain, proglucagon is processed by PC1/3 into oxyntomodulin, intervening peptide-2 (IP-2), GLP-1, and GLP-2. GLP-1 is degraded by DPP-4 via cleavage of two amino acids from the N terminus, or by NEP-24.11 through cleavage of the C terminus in vivo. The cleaved products are eventually eliminated in the kidney. UTR, untranslated region. enhance insulin secretion after a mixed meal, but GLP- hormone, in addition to epinephrine, and its release is 1 is more potent than GIP (Nauck et al., 1993b; Elahi reciprocally correlated with insulin secretion in glucose et al., 1994). In the human circulation, GIP concentra- oscillation to mobilize hepatic glucose in the fasting tion is eightfold higher than GLP-1; in type 2 diabetic state, thereby helping to ensure the maintenance of patients, GLP-1 has more activity than GIP, but their normoglycemia. In T2DM, both fasting hyperglucago- effects on insulin secretion seem to be additive (Nauck nemia and exaggerated glucagon responses, which most et al., 1993a, 2004; Elahi et al., 1994). likely contribute to the of patients, were 1. Effect on Glucose Homeostasis. The insulinotropic observed (Shah et al., 2000; Toft-Nielsen et al., 2001). In- activity of GLP-1 is strictly glucose-dependent mediated terestingly, only GLP-1, but not GIP, inhibits glucagon through its receptor at the membrane of pancreatic b secretion (Nauck et al., 1993b). However, the detailed cells (Kreymann et al., 1987; Mojsov et al., 1987; Holst, mechanism(s) by which GLP-1 suppresses glucagon secre- 2007). GLP-1 could not stimulate insulin secretion at low tion remains unclear. Because the levels of GLP-1 mRNA levels of glucose in humans (Kreymann et al., 1987). detected in a cells varied between none and 20% of a cell GLP-1R belongs to the class B GPCR subfamily whose population, it is generally thought that local elevated members include receptors for peptidic hormones such as insulin and somatostatin in response to GLP-1 stimulation glucagon, secretin, GIP, etc. The binding of GLP-1 to its are capable of suppressing glucagon secretion in a cells receptor activates heterotrimeric Gs protein, which sub- (Orskov et al., 1988; Heller et al., 1997; de Heer et al., 2008; sequently elicits adenylate cyclase activity, resulting Godoy-Matos, 2014). Nonetheless, in type 1 diabetic pa- in cAMP formation (Gromada et al., 1998, 2004). The tients with absent b cell activity who lack insulin and increased level of cAMP in turn leads to activation of somatostatin, GLP-1 could still reduce glucose concentra- protein kinase A (PKA) and the cAMP-regulated guanine tions, suggesting a direct suppression of glucagon secretion nucleotide exchange factor II (Ozaki et al., 2000). In the (Creutzfeldt et al., 1996; Gromada and Rorsman, 2004). presence of high levels of glucose, GLP-1 has an effect on 2. Effect on Gastric Emptying. GLP-1 also has an ATP-sensitive (KATP) or voltage-gated potassium and important inhibitory activity on gut motility and gas- calcium channels, resulting in membrane depolarization trointestinal secretion (Wettergren et al., 1993; Nauck and Ca2+ release from both internal and extracellular et al., 1997). It not only inhibits meal-induced pancre- stores. Increased Ca2+ together with cAMP will then pro- atic secretion, but also the gastric emptying process in mote exocytosis of vesicles containing insulin (Prentki and humans (Wettergren et al., 1993). Its suppression of Matschinsky, 1987; Renstrom et al., 1997). This glucose- -induced acid secretion was demonstrated by dependent insulinotropic action of GLP-1 involves the injection of GLP-1 and/or peptide YY. Both peptides are glucose transporter, metabolic ADP/ATP ratio, KATP in- released from L cells in the ileal mucosa of healthy hibition, Ca2+ channel opening, and, ultimately, insulin people (Wettergren et al., 1997) and can exhibit additive secretion (Gromada et al., 2004; Dyachok et al., 2006). effects on gastrin-stimulated acid secretion, a function Another major activity of GLP-1 to reduce blood of unabsorbed nutrients in the ileum (Holst, 1997). It glucose relates to the suppression of glucagon secretion was subsequently shown that this inhibitory action of from a cells of the endocrine pancreas (Gromada and GLP-1 is mediated via a vagal pathway (Wettergren Rorsman, 2004). Glucagon is a major hyperglycemic et al., 1994). This ileal-brake activity of GLP-1 was Glucagon-Like Peptide-1 and Its Receptors 959 further demonstrated using the GLP-1R antagonist hydrogen peroxide stress, implying an unconventional exendin9–39, and therefore, is believed to have phys- action of GLP-1 (Ban et al., 2010). In a rat ischemia- iologic relevance (Schirra et al., 2006; Maljaars et al., reperfusion model using isolated perfused and 2008). whole animal, GLP-1 significantly decreased infarction 3. Effect on Food Intake. Another physiologic func- size, and this protection was abolished by exendin9–39, tion of GLP-1 concerns inhibition of food intake that as well as inhibitors of , phosphatidyl- may have therapeutic value for body weight reduction. inositol 3-kinase (PI3K), and p42/44 mitogen-activated Whether this is related to its ileal brake effect is still protein kinase (MAPK), suggesting that these pathways debated. At least two neural mechanisms, central and were involved in GLP-1–mediated cardio-protection peripheral, are involved in GLP-1 suppression of appe- (Bose et al., 2005a). A direct action of GLP-1 on myo- tite and food intake. GLP-1 is expressed in the neurons cardial contractility and glucose uptake in normal and of the brain stem, and GLP-1R is present in the hypo- postischemic isolated rat was also observed thalamic areas that control energy homeostasis and (Zhao et al., 2006). GLP-1 treatment significantly food intake, including the arcuate nucleus, paraven- increased glucose uptake and decreased left ventricu- tricular nucleus, and dorsomedial nucleus (Jin et al., lar end-diastolic and developed pressures. Infusion of 1988; Kanse et al., 1988; Zheng et al., 2015). Intra- GLP-1 into live dogs increased myocardial glucose cerebroventricular injection of GLP-1 inhibits food in- uptake, which could be blocked by p38 MAPK kinase take in rats, and this activity is blocked by exendin9–39 or endothelial synthase inhibitors, point- (Tang-Christensen et al., 1996; Turton et al., 1996), or ing to a direct effect of GLP-1 in myocardium (Nikolaidis by the arcuate nucleus-damaging reagent, monosodium et al., 2004a; Bhashyam et al., 2010). Meanwhile, GLP- glutamate (Tang-Christensen et al., 1998). In contrast, 1R was detected in human coronary artery endothelial GLP-1 released by L cells of the intestine after a meal cells and umbilical vein endothelial cells, and GLP-1 or inhibits gut mobility and gastric emptying, allowing exendin-4 treatment led to nitric oxide production in nutrients in the ileum to reduce food intake (Read et al., both cell types, indicative of GLP-1 involvement in the 1994). Indeed, infusion of GLP-1 into normal human vasculature (Nystrom et al., 2004; Erdogdu et al., 2010; subjects significantly enhances satiety and decreases Ishii et al., 2014). GLP-1 infusion into rats also increased food intake (Flint et al., 1998). Consistent findings have heart rate and , thereby reflecting its shown that GLP-1R agonism promotes weight loss and direct role in the heart (Barragan et al., 1996). In recent improves glucose homeostasis in rodents, monkeys, and clinical trials with either GLP-1R agonists or DPP-4 humans (Verdich et al., 2001; Barrera et al., 2011), inhibitors, general beneficial effects of GLP-1 on the and such weight-reducing properties have also been cardiovascular system in both normal and diabetic well-documented for two marketed GLP-1 mimetics, subjects have been revealed, an interesting finding that exenatide (exendin-4) and (Moretto et al., may lead to potential new treatment of cardiovascular 2008; Astrup et al., 2009; Norris et al., 2009; Lean et al., diseases, although these effects require a long-term 2014). Although the exact mechanism mediating re- validation (van Genugten et al., 2013; Avogaro et al., duced food intake by peripherally administered GLP-1 2014; Ussher and Drucker, 2014; see V. Pharmaceutical has yet to be elucidated, it may involve signals gener- Development and Therapeutics). ated by GLP-1 binding to its receptors on neurons in the 5. Effect on Immune Response. GLP-1 can also regulate or hepatoportal bed (Burcelin immune responses. Its receptor mRNA was discovered et al., 2001; Holst, 2007). in multiple immune cell types from mice, including 4. Effect on Cardiovascular Activity. Recently, there thymoyctes, splenocytes, bone marrow–derived cells, is increasing evidence suggesting that GLP-1 may play regulatory T cells, macrophages, and invariant natural a crucial role in the cardiovascular system (Grieve et al., killer T cells (Hadjiyanni et al., 2010; Hogan et al., 2009; Ussher and Drucker, 2014). GLP-1R is widely 2011; Panjwani et al., 2013). Liraglutide treatment of expressed in the heart and blood vessels, such as patients with psoriasis, an inflammatory condition vascular , cardiomyocytes, endocardium, associated with metabolic diseases such as obesity, and coronary /smooth muscle, in both diabetes, and dyslipidemia, led to improvement of rodents and humans (Campos et al., 1994; Wei and psoriasis area and severity index as well as decreased Mojsov, 1995; Bullock et al., 1996). In an early study, secretion from invariant natural killer T cells treatment of adult rat cardiac myocytes with GLP-1 in a glycemic control-independent manner (Hogan increased cAMP levels, but did not lead to increased et al., 2011). High-fat diet–fed mice treated with cardiomyocyte contractility, as would be anticipated in exendin-4 displayed decreased mRNA levels of the the heart (Vila Petroff et al., 2001). Interestingly, proinflammatory monocyte chemoattractant treatment of mouse cardiomyocytes with GLP-19–36 amide protein 1, -a, and signal transducer (a GLP-1 degradation product in vivo) resulted in Akt and activator of transcription 3 (Koehler et al., 2009), and, activation, extracellular regulated kinase (ERK) phosphor- in a type 1 diabetes animal model in which islets were ylation, and reduced apoptosis induced by hypoxia or transplanted into nonobese diabetic mice, GLP-1/gastrin 960 de Graaf et al. treatment increased the number of transforming growth C. Structure – – factor-b1 secreting lymphocytes and decreased IFN-g Structurally, class B GPCRs consist of a large N-terminal secreting lymphocytes with delayed onset of diabetes extracellular domain (ECD) and a seven-transmembrane (Suarez-Pinzon et al., 2008). (7TM) helix domain, comprising the GPCR signature of 6. Effect on Kidney Function. Some experimental data seven membrane spanning a-helices [transmembrane point to the participation of GLP-1 in kidney function. (TM)1–7], connected by three extracellular (ECL) and Infusion of GLP-1 into healthy and obese human subjects intracellular (ICL) loops, and a C-terminal helix 8 enhanced sodium , urinary secretion, and glo- (Schioth et al., 2003; Hollenstein et al., 2014). Pharma- merular filtration rate, suggesting a renal protective effect cological studies with truncated, chimeric, and mutated of this peptide (Gutzwiller et al., 2004). In rats, GLP-1 was ligand and receptor variants, together with peptide able to downregulate Na+/H+ exchanger isoform 3 (NHE3) ligand-bound ECD crystal structures and the first 7TM in the renal proximal tubule, implying a potential thera- crystal structures of class B GPCRs, have provided the peutic value for and disorders of sodium basis for a two-domain–binding mechanism of peptide retention (Crajoinas et al., 2011). Rats administered hormone ligands to secretin-like class B GPCRs (Parthier with exendin-4 significantly improved renal function et al., 2009; Donnelly, 2012; Hollenstein et al., 2014). and reduced inflammation, proteinuria, and fibrosis in According to this peptide ligand-binding mechanism: 1) the kidney via a mechanism that was independent of the C terminus of the peptide ligand forms an initial glucose lowering (Kodera et al., 2011). complex with the ECD, and this allows 2) the N terminus 7. Effect on Nervous System. In addition to the of the peptide ligand to interact with the 7TM domain metabolic function in the brain, GLP-1 may also exert (7TMD) and activate the class B GPCR to couple to G neuroprotective and neurotropic effects (Heppner and and other effectors to mediate intracellular Perez-Tilve, 2015). The aggregation of amyloid b pro- tein (Ab) and the microtubule-associated protein Tau signaling processes (see III. Glucagon-Like Peptide-1 – cause senile plaques and neurofibrillary tangles, result- Receptor). This section gives an overview of the structure ing in loss of long-term potentiation, one of the major activity relationship (SAR) of GLP-1 peptides, thereby characteristics of Alzheimer’s disease (AD). Infusion of providing important information regarding the molec- GLP-1 or exendin-4 into the lateral ventricles of mice ular determinants of ligand binding and functional decreased endogenous level of Ab,andtreatmentof activities at the GLP-1R. The development of GLP-1 rat hippocampus neurons with GLP-1 and exendin-4 analogs is described in detail in V. Pharmaceutical prevented Ab-induced cell death (Perry et al., 2003). Development and Therapeutics, whereas SAR of GLP- Intracerebroventricular dosing of GLP-1 enhanced syn- 1R will be discussed explicitly in III. Glucagon-Like aptic plasticity in the hippocampus and completely Peptide-1 Receptor. Alignments of the sequences and reversed impairment in long-term potentiation caused structures of GLP-1 with GLP-2, glucagon, GIP, and by subsequent injection of Ab (Gault and Holscher, exendin-4 are presented in Fig. 2. This also annotates 2008). GLP-1R was found in nigrostriatal neurons, and the structural properties of GLP-1, effects of GLP-1 loss of these neurons is a feature of Parkinson’s disease studies, and GLP-1R and exendin-4 interac- (PD). GLP-1 or exendin-4 supported cell viability during tion sites in the corresponding GLP-1R ECD crystal hypoxic injury in primary neurons from rat cerebral structures (Runge et al., 2008; Underwood et al., 2010). Peptide ligand residues shown are annotated as three- cortical tissues; this activity was blocked by GLP-19–36 (a GLP-1R antagonist) and not observed in neurons letter codes with residue number as super- 7 from GLP-12/2 mice (Li et al., 2009). Exendin-4 also scripts (e.g., His , histidine at position 7), whereas receptor maintained cell viability and reduced apoptosis caused residue numbers are annotated as single-letter codes, by H2O2-induced oxidative stress in NSC19 neuronal Uniprot numbers, and Ballesteros-Weinstein/Wootten cells, a spinal cord cell line with similarities to cells in numbers/secondary structure motif as superscripts, ac- the central nervous system (CNS) (Li et al., 2012b). cording to IUPHAR guidelines (Pawson et al., 2014) and Obviously, GLP-1 and GLP-1R are widely expressed class B GPCR residue-numbering guidelines (Wootten in many tissues, and their physiologic actions have et al., 2013c; Isberg et al., 2015), respectively. According been incrementally elucidated, especially after the to the Ballesteros–Weinstein class A GPCR (Ballesteros extensive clinical application of GLP-1 mimetics, in- and Weinstein, 1995) and Wootten class B GPCR (Wootten cluding GLP-1R agonists and DPP-4 inhibitors, in the et al., 2013c) residue-numbering schemes, the single most last decade. Although its roles in some tissues, such as conserved residue in each TM helix is designated X.50 adipocytes or skeletal muscles, are still illusive, GLP- (Ballesteros-Weinstein number used for comparison within 1–related therapies have clearly provided multiple class A GPCRs as well as to compare across GPCR classes) benefits to millions of patients suffering from meta- or X.50b (Wootten number for comparison with class B bolic disorders, and such effects are predominantly, GPCRs). X is the TM helix number, and all other residues but perhaps not always, based on signaling pathways in that helix are numbered relative to this conserved mediated by its receptor. position (Hollenstein et al., 2014). GLP-1R residues that Glucagon-Like Peptide-1 and Its Receptors 961

Fig. 2. Structural characteristics of GLP-1 and its cognate receptor. (A) GLP-1–bound full-length GLP-1R homology model based on a previously published full-length glucagon-bound GCGR model combining structural and experimental information from the GCGR 7TMD crystal structure (PDB: 4L6R), the GCGR ECD structure (PDB: 4ERS), and the ECD structure of GLP-1–bound GLP-1R (PDB: 3IOL), complemented by site-directed mutagenesis, electron microscopy, hydrogen-deuterium exchange, and cross-linking studies (Siu et al., 2013; Yang et al., 2015b, 2016). The C-terminal helix of GLP-1 bound to the ECD region of GLP-1R is depicted as cartoon, whereas the atoms of the flexible N-terminal region of GLP-1 predicted to be bound to the 7TMD of GLP-1R are depicted as spheres. GLP-1 is color coded according to mutation effects (blue: ,fourfold effect, orange: 4- to 10-fold effect, red: .10-fold effect IC50; see II. Glucagon-Like Peptide-1); mutation effects of GLP-1R are reported in Fig. 3 and Table 1. The Ca/Cb atoms of GLP-1/GLP-1R residue pairs identified in photo cross-linking studies (Chen et al., 2009, 2010b; Miller et al., 2011) are depicted as green-colored spheres. (B) Structural alignment of the ECD structures of GLP-1 and exendin9–39–bound GLP-1R (PDB: 3IOL, 3C59), GIP-bound GIPR (PDB: 2QKH), and the mAb23-bound GCGR ECD structure (PDB: 4ERS). Comparison of the crystal structure binding modes of (C) GLP-1 and (D) exendin9–39. The surfaces of GLP-1R residues involved in important apolar interactions with GLP-1/apolar are colored pink, whereas residues involved in polar interactions described in II. Glucagon-Like Peptide-1 are also depicted as sticks (and their H-bond interaction networks are depicted as dashed lines). (E) Structure-based sequence alignment of GLP-1, exendin9–39, glucagon, GIP, and GLP-2. The regions of the peptide ligands solved in ECD–ligand complex crystal structures are marked above the amino acid sequences using the same color coding as in (B). Amino acids of GLP-1 are marked according to mutation study effects, as indicated in (A). The residues that are boxed are found in an a-helical conformation in the crystal structure complex (solid lines: GLP-1, exendin9–39, GIP) or in NMR studies in micelle DPC (dashed lines: glucagon, GLP-2), as described in II. Glucagon-Like Peptide-1. 962 de Graaf et al. are most conserved in secretin-like class B GPCRs are demonstrated that exendin-4 has a higher a-helical S1551.50b, H1802.50b,E2473.50b,W2744.50b,N3205.50b, propensity than GLP-1 in solution (Runge et al., 2007). G3616.50b, and G3957.50b. GLP-1 and glucagon peptide Combination of these biophysical data with pharmaco- ligands start with amino acid residue 7 (His7) due to post- logical studies showed that there is a positive correla- translational processing (Fig. 2), whereas the homolo- tion between the stability and a-helical propensity of gous GLP-2, GIP, and exendin-4 start with His1 and the ligand in solution and its affinity for the ECD of Tyr1, respectively. GLP-1R (Runge et al., 2007). Comparison of the crystal 1. a-Helical C-Terminal Region. NMR, circular di- structures of GLP-1–bound and exendin9–39-bound chroism (CD) spectroscopy, and X-ray crystallography ECD provides possible explanations for the higher studies show that GLP-1 is unstructured in aqueous stability of the a-helix of exendin-4 as opposed to GLP-1 solution, but adopts a helical structure in a membrane- (Runge et al., 2008; Underwood et al., 2010). Although like environment and by binding to its receptor (Thornton the a-helix of exendin-4 is stabilized by strong intra- and Gorenstein, 1994; Neidigh et al., 2001; Runge et al., molecular ionic interactions between Glu16/Glu17 and 2007; Underwood et al., 2010). This conformational Arg20 and between Glu24 and Lys27, corresponding change upon receptor binding is also proposed for other (Gln23, Lys26) or differentially positioned (Glu27, Lys34) class B GPCR peptide ligands, including glucagon polar residues in GLP-1 do not form such intramolecu- (Braun et al., 1983; Siu et al., 2013), GIP (Alana et al., lar interactions when bound to the isolated ECD of 2006; Parthier et al., 2007), and exendin-4 (Runge et al., GLP1-R [ (PDB): 3IOL]. The GIP- 2007, 2008) (Fig. 2), implying a conserved receptor- bound GIP receptor (GIPR) ECD crystal structure ligand–binding mechanism (Parthier et al., 2009). indicates that the a-helix of GIP is stabilized by similar Two-dimensional NMR experiments indicate that the (Asp15-Gln19) and alternative (Gln20-Asn24) intramolec- C-terminal region of GLP-1 (Thr13-Lys34)isinana-helical ular H-bond interactions (Parthier et al., 2007), whereas conformation in trifluorethanol and dodecylphosphocholine glucagon may also be able to form helix-stabilizing (DPC) micelles, with a less well-defined a-helical region intramolecular H-bond networks (e.g., Asp21-Arg24, around Gly22 (Thornton and Gorenstein, 1994; Neidigh Arg23-Asp27) (Siu et al., 2013). In addition to the higher et al., 2001; Underwood et al., 2010). NMR studies a-helix propensity, the more pronounced amphiphilic suggest that exendin-4 adopts a more well-defined nature of exendin-4 enables stronger polar and hydro- single helix (Ser11-Lys27) than GLP-1 in DPC micelles phobic interactions with the ECD of GLP-1R via (Neidigh et al., 2001). These NMR data are in line with opposite sides of the a-helix than GLP-1 (vide infra). ligand-bound GLP-1R ECD crystal structures showing Moreover, exendin-4 is eight residues longer than GLP-1, that the GLP-1 is a kinked but continuous a-helix and NMR studies have shown that this extended 13 33 32 39 (Thr –Val ), whereas the truncated exendin9–39 is a C-terminal region, comprising Ser -Ser ,formsa straighter helix (Leu10-Lys27) than GLP-1 when bound stable tertiary structure that folds around Trp25 in to GLP-1R (Runge et al., 2008; Underwood et al., 2010). trifluorethanol and glycol (Neidigh et al., 2001). This In the ECD-bound crystal structure, the C-terminal Trp-cage conformation is, however, not observed in a-helix of GLP-1 (Ala24-Val33) is stabilized by interac- NMR studies in DPC micelles (Neidigh et al., 2001) and tion with the ECD of GLP-1R, whereas the N-terminal has weak electron density in the exendin9–39-bound helix (Thr13-Glu21) does not interact with the ECD. ECD GLP-1R crystal structure (Runge et al., 2008), Although it cannot be excluded that the kink in GLP-1 suggesting that a stable Trp-cage conformation (which observed in the crystal structure is a result of crystal is absent in GLP-1) is only partially populated in the packing between the N-terminal part of GLP-1 (Gly10- receptor-bound state of exendin-4. Glu21) and symmetry-related ECDs, the presence of 2. Flexible N-Terminal Region. NMR and X-ray crys- two helical segments is consistent with the SAR stud- tallography studies indicate that the N-terminal regions ies with conformationally constrained GLP-1 analogs preceding the conserved a-helix of GLP-1 (His7-Thr13), (Miranda et al., 2008; Murage et al., 2008). Cyclization exendin-4 (His1-Thr7), GIP (Tyr1-Ile7), glucagon (His7- of (mutated) residues Lys16-Glu20 and Met18-Ala22 by a Thr13), and other class B GPCR peptide ligands are lactam bridge (constraining these regions in an a-helical flexible in solution as well as in membrane-bound and conformation) does not affect binding affinity and activity, ligand-bound states (Braun et al., 1983; Neidigh et al., whereas cyclization of residues Thr11-Asp15 even im- 2001; Parthier et al., 2007; Runge et al., 2008; Underwood proved potency for GLP-1R compared with the corre- et al., 2010). The structure of the N-terminal region of sponding linear analogs (Miranda et al., 2008; Murage GLP-1 (His7-Thr13) was not clearly elucidated in NMR et al., 2008). studies in DPC micelles due to high conformational Characterization of the stability and conformational flexibility (Neidigh et al., 2001). In the ECD-bound GLP-1 changes of full-length, truncated, and GLP-1/exendin-4 crystal structure, this N-terminal region is unstructured, chimeric peptides upon binding to the isolated ECD and no electron density was observed for His7-Glu9 of GLP-1R by far-UV CD, differential scanning calo- (Underwood et al., 2010). Whereas the GLP-1R ECD rimetry, and fluorescence spectroscopy measurements crystal structures provide atomic details of the molecular Glucagon-Like Peptide-1 and Its Receptors 963 interactions of the C-terminal regions of GLP-1 and conserved structural fold and binding orientation sug- exendin9–39 with the ECD of GLP-1R (Runge et al., gest that common mechanisms underlie ligand recogni- 2008; Underwood et al., 2010), these structures do not tion of class B GPCRs and indicate that peptide ligand give information on the receptor-bound conformation of selectivity is in part determined by specific interac- the flexible ligand N terminus, nor on its interactions tions of the C-terminal a-helix of the ligand with the with GLP-1R. Ligand and receptor mutation studies ECD. The ligand-binding mode observed in the GLP-1R suggest that an extended flexible conformation of these crystal structure is consistent with GLP-1 (vide infra) first residues allows the peptide ligands of class B GPCRs and GLP-1R (see III. Glucagon-Like Peptide-1 Receptor) to reach deep into the pocket (Hollenstein et al., 2014). mutation studies, as well as photo cross-linking experi- This receptor-bound peptide conformation is proposed to ments placing Gly35 located in the a-helix of GLP-1 in be stabilized by an amino acid motif (Thr11-Phe12-Thr13 close proximity to E125ECD located in the linker region in GLP) that is conserved in class B GPCR ligands between the ECD and 7TMD of GLP-1R (Chen et al., (Neumann et al., 2008), which can induce an N-capping 2009). The structural details of the ECD and 7TMD of conformation similar to the one observed in the receptor- GLP-1R are discussed in detail in III. Glucagon-Like bound NMR structure of pituitary adenylate cyclase– Peptide-1 Receptor. activating polypeptide (PACAP) (Inooka et al., 2001). The a-helices of GLP-1, exendin-4, glucagon, and GIP Substituting three residues in the flexible N terminus of are amphiphilic, containing a hydrophilic and hydro- GLP-1 by corresponding PACAP residues (Ala8Ser/ phobic region at opposite sides of the helix. Conserved Glu9Asp/Thr11Ile) does not affect affinity and potency apolar residues in the C-terminal part of GLP-1 (Phe28, for GLP-1R (Xiao et al., 2001), implying that GLP-1 Ile29, Leu32), exendin-4 (Phe22, Ile23, Leu26), GIP (Phe22, adopts a similar conformation to PACAP upon binding Val23, Leu26), and glucagon (Phe28, Val29, Leu32) share to its receptor. Recently, NMR structures of an 11-mer a similar hydrophobic interaction site with the ECD GLP-1 analog were solved in alterative conformations of the corresponding receptor (GLP-1R/GIPR/GCGR: containing a C-terminal a-helix (PDB: 2N08) and an L32ECD/A32/M29, T35ECD/L35/L32, V36ECD/Y36/F33, and ECD ECD N-terminal b-turn (PDB: 2N09), and stabilization of these W39 /39/36 in a1, Y69 /68/65 in b-turn 1 connect- ECD ECD ECD conformations by cyclization cross-links (PDB: 2N0N and ing b1-b2, Y88 /87/84, L89 /88/85, and P90 /89/ 2N0I) differentially influenced GLP-1R–binding affinity 86 in b-turn 2 connecting b3-b4) (Parthier et al., 2007; and potency (Hoang et al., 2015). The accumulated Runge et al., 2008; Underwood et al., 2010; Koth et al., ligand and receptor SAR (see II. Glucagon-Like Peptide-1 2012). Differences in hydrophobic/polar interactions and III. Glucagon-Like Peptide-1 Receptor)suggeststhata with the N-terminal part of the ligand a-helix as well flexible conformation of the first seven residues allows as ligand-specific ionic interactions with the ECD can GLP-1 to interact with residues in the 7TM-binding pocket partially explain the different relative affinities of of GLP-1R, and that the ligand N terminus may adopt a GLP-1, exendin-4, GIP, and glucagon for the ECD of more constrained conformation to activate the receptor. the corresponding receptor. The positively ionizable Lys26 20 3. Interaction between the C-Terminal Helix and of GLP-1 and homologous Arg of exendin9–39 both form ECD. Comparison of the crystal structures of GLP-1 ionic interactions with E128ECD in the C-terminal region 27 and exendin9–39-bound GLP-1R ECD (Runge et al., of the ECD of GLP-1R. The Lys residue of exendin9–39 2008; Underwood et al., 2010), GIP-bound GIPR ECD forms an additional ionic interaction with E128ECD in (Parthier et al., 2007), and the antibody bound glucagon GLP-1R (Runge et al., 2008; Underwood et al., 2010), receptor (GCGR) ECD (Koth et al., 2012) provides which in combination with the higher a-helical pro- information about similarities and differences in class pensity of exendin-4 compared with GLP-1 (Runge B GPCR-bound peptide ligand conformations, and gives et al., 2007) might explain the increased affinity of insights into the structural determinants of class B exendin-4/exendin9–39 for the isolated ECD of GLP-1R GPCR ligand recognition and selectivity (Fig. 2). The compared with GLP-1. There is no ionic interaction crystal structures of the ECDs of different class B between peptide ligand and ECD side chains observed GPCRs show that this domain has a conserved fold, in the GIP-bound GIPR ECD crystal structure and the including two central antiparallel b-sheets (b1-b4) and ECD GCGR-glucagon docking model based on the an N-terminal a-helix (a1) interconnected by several antibody-bound GCGR and GLP-1–bound GLP-1R crystal loops and stabilized by three conserved disulfide bonds structures. It should be noted, however, that the apolar (Donnelly, 2012; Pal et al., 2012; Hollenstein et al., residues of GLP-1 (Val33), GIP (Leu27), and glucagon 33 27 2014) (for more detailed description of the ECD of GLP- (Met ) that are aligned with Lys of exendin9–39 form 1R, see III. Glucagon-Like Peptide-1 Receptor). Class B an additional hydrophobic interaction site with the ECD GPCR ECD peptide ligand complexes exhibit overall a of GLP-1 (Y69ECD/L123ECD), GIPR (Y68ECD /H115ECD), similar binding mode in which the C terminus of the and GCGR (Y65ECD/A118ECD), respectively. Ala24 and peptide ligand adopts an a-helical conformation that Ala25 of GLP-1 and Val19 in exendin-4 form another binds between a1 and b1–b4 of the ECD (Parthier et al., hydrophobic interaction surface with a1 of the ECD of 2009; Donnelly, 2012; Hollenstein et al., 2014). The GLP-1R. The structurally aligned Gln19 of GIP forms 964 de Graaf et al. an H-bond network with Asp15 and the N-terminal example, unnatural amino acid substitution and conju- ECD ECD backbone of a1 of the GIPR ECD (Q30 and A32 ), gation (Manandhar and Ahn, 2015), is discussed in V. whereas corresponding residues in glucagon (Arg24, Pharmaceutical Development and Therapeutics. Ala25) may form a polar/apolar interaction site with the 1. Truncated GLP-1 Analogs. N-terminally trun- 7 N-terminal region of the a1 helix of GCGR (Fig. 2). cated forms of GLP-1 (including des-[His ]-GLP-18–36 7 8 4. Interaction between Flexible N terminus and and des-[His ,Ala ]-GLP-19–36) are competitive antag- 7TMD. The peptide ligand-bound crystal structures onists of GLP-1R, whereas C-terminally truncated 37 36 of the ECDs of class B GPCRs (including GLP-1– and GLP-1 constructs (including des-[Gly ,Arg ]-GLP-17–37) exendin-4–bound GLP-1R, GIP-bound GIPR, and remain agonists (Mojsov, 1992; Montrose-Rafizadeh antibody-bound GCGR) do not provide information et al., 1997). The binding affinities of N-terminally about the molecular interactions between the receptor truncated GLP-1 constructs are not affected by muta- and the flexible N-terminal region of peptide ligands. tions in the 7TMD of GLP-1R (Al-Sabah and Donnelly, Receptor/ligand mutagenesis and photo cross-linking 2003a,b; López de Maturana et al., 2004), indicating studies nevertheless indicate that the 7TMD of class B that interactions between the N-terminal region of GLP-1 GPCRs determines binding (selectivity) of the flexible and the 7TMD of GLP-1R are required for receptor N-terminal region of peptide ligands (Donnelly, 2012; activation. Most truncated GLP-1 constructs have a Pal et al., 2012; Hollenstein et al., 2014). A recently decreased affinity for GLP-1R (Mojsov, 1992; Montrose- reported full-length GCGR–glucagon model based on Rafizadeh et al., 1997; Donnelly, 2012), suggesting that the crystal structures of the GCGR 7TMD (Siu et al., interactions with both the ECD and the 7TMD are 2013), the antibody-bound GCGR ECD (Koth et al., important determinants of GLP-1 binding. Similar SARs 2012), the GLP-1–bound GLP-1R ECD (Underwood have been observed for the endogenous ligands of related et al., 2010), and the N-capped conformation of PACAP class B GPCRs, including glucagon/GCGR (Unson et al., (Inooka et al., 2001) offers a template for full-length 1989) and GIP/GIPR (Hinke et al., 2001), demonstrating GLP-1–bound GLP-1R models (Fig. 2A) that is consis- that interactions of peptide ligands in the 7TMD of tent with the results of mutation studies of GLP-1 (vide class B GPCRs are required for receptor activation. 7 infra), GLP-1R (see III. Glucagon-Like Peptide-1 Recep- The N-terminally truncated des-[His ]-GLP-18–36 and 7 8 tor), and other class B GPCRs (Hollenstein et al., 2014). des-[His ,Ala ]-GLP-19–36 variants of GLP-1 have a This full-length GLP-1R model furthermore satisfies 100- and 1000-fold lower affinity than that of wild- spatial constraints defined by GLP-1R cross-linking stud- type, whereas truncation of more than two N-terminal ies connecting the following: 1) Ala24 in the C-terminal residues further diminishes GLP-1R binding (Mojsov, part of the a-helix of GLP-1 to E133ECD in the region 1992; Montrose-Rafizadeh et al., 1997). Truncation of linking the ECD and 7TMD of GLP-1R (not solved in the up to three C-terminal residues (Gly35, Arg36, and Gly37) GLP-1R crystal structure); 2) Leu20 in the N-terminal part only has a moderate effect on GLP-1R–binding affinity of the a-helix of GLP-1 to W297ECL2 in ECL2 between (up to fivefold decrease), but further deletion of C-terminal TM4 and TM5 in GLP-1R; 3) Phe12 and Val16 in the residues decreases binding affinity significantly. Never- flexible N-terminal region of GLP-1 that are positioned theless, several undecapeptide GLP-17–15 analogs in near Y1451.40b and L1411.36b in the TM1 of GLP-1R, which the C-terminal 21 residues are replaced with a respectively; and 4) a photolabile probe at position biphenylalanine dipeptide have been reported to pos- 6 (one position before His7) in GLP-1 to Y205ECL1 in sess almost the same potency as wild-type GLP-1 ECL1betweenTM2andTM3inGLP-1R(Chenetal., (Mapelli et al., 2009). 2009, 2010b; Miller et al., 2011) (Fig. 2A). In contrast to GLP-1, which requires its N-terminal region for high-affinity binding to GLP-1R, the homol- D. Mutagenesis ogous agonist exendin-4 and the N-terminally trun- The molecular determinants of GLP-1R ligand bind- cated antagonist exendin9–39 have similar affinities for ing and functionality have been investigated exten- GLP-1R (Montrose-Rafizadeh et al., 1997). Although sively using truncated, chimeric, and site-specifically full-length GLP-1R binds exendin-4 and GLP-1 with substituted GLP-1, exendin-4, and peptide ligands of similar high affinity, the isolated ECD maintains high closely related class B GPCRs (including glucagon and affinity for exendin-4 and exendin9–39, but has de- GIP). This section gives an overview as to how these creased affinity for GLP-1 (Al-Sabah and Donnelly, data complement the structural information on GLP-1 2003a; López de Maturana et al., 2003). In addition, (and the ECD–GLP-1 complex) described above. It GLP-1 binding is more sensitive to site-directed muta- should be noted that the current overview only focuses genesis of the 7TMD of GLP-1R than exendin-4 (Al- on changes to natural amino acids, as the incorporation Sabah and Donnelly, 2003a,b; López de Maturana et al., of photoactive labels in GLP-1 for GLP-1/GLP-1R cross- 2003; see III. Glucagon-Like Peptide-1 Receptor). Con- linking studies (Chen et al., 2009, 2010b; Miller et al., sistently, radioligand competition studies combining 2011) is covered in this section, whereas the develop- isolated C-terminal (ECD) and N-terminal (7TMD) ment of GLP-1 analogs by other modifications, for GLP-1R constructs with native and N-terminally or Glucagon-Like Peptide-1 and Its Receptors 965

C-terminally truncated GLP-1 and exendin-4 showed and GLP-1 (Runge et al., 2003b). The chimeric GLP-1- that: 1) GLP-1 binding is primarily determined by (His7-Leu20)/glucagon-(Asp21-Thr35) is unable to bind interactions with the ECD, but also requires interac- and activate GCGR, but its binding affinity and potency tions with the 7TMD of GLP-1R; and 2) exendin-4– are rescued by substituting the 7TMD of GCGR with the binding affinity is mainly determined by interactions 7TMD of GLP-1R, suggesting that the N-terminal with the ECD of GLP-1R and does not heavily depend on region of GLP-1 (His7-Leu20) interacts with the 7TMD interactions with the 7TMD (López de Maturana et al., of GLP-1R (Runge et al., 2003b). The GCGR(ECD)/ 2003). Based on pharmacological studies with truncated GLP-1R(7TM) chimera has equal binding affinity and GLP-1 and exendin-4, it was further postulated that the potency for GLP-1 and the chimeric glucagon-(His7- eight-residue C-terminal extension of exendin-4 (Trp-cage, Leu20)/GLP-1-(Asp21-Gly37), although these are de- see above) may play a role in its superior affinity for the creased compared with wild-type GLP-1R, indicating ECD of GLP-1R (Al-Sabah and Donnelly, 2003a). that the ECD of GLP-1R is the major determinant of 2. Chimeric GLP-1 Analogs. Chimeric constructs of GLP-1/glucagon selectivity by interacting with the GLP-1 in combination with other class B GPCR pep- C-terminal region of GLP-1. Chimeras combining tide ligands (including exendin-4, glucagon, GIP, and different N-terminal and C-terminal regions of GLP-1 PACAP) have been used to identify structural determi- (His7-Leu20,Asp21-Arg36)andGIP(His1-Leu14,Asp14- nants of ligand selectivity for GLP-1R (and other class B Lys30), or substituting the middle parts of GIP with GPCRs). Radioligand competition studies with trun- corresponding residues of GLP-1 (Ser18-Ala24,Glu21- cated and chimeric GLP-1 and exendin-4 peptides Ala24), all had more than 100-fold lower binding affinity suggested that divergent residues in the central part of for GLP1-R compared with GLP-1-(His7-Arg36) (Hareter GLP-1 (Val16-Arg36) and exendin-4 (Leu10-Gly30) de- et al., 1997). Substituting three to five residues in the termine GLP-1/exendin-4 selectivity for the ECD of N-terminal part of GLP-1 by corresponding resi- GLP-1R, and indicated that the Trp-cage only plays a dues from GIP (His7Tyr/Thr13Ile/Val16Tyr), secretin minor role in the increased affinity of exendin-4 for the (Ala8Ser/Glu9Asp/Asp15Glu/Val16Leu), vasoactive intes- ECD of GLP-1R compared with GLP-1 (Runge et al., tinal peptide (Ala8Ser/Glu9Asp/Gly10Ala/Thr11Val), or 2003b). These pharmacological data are consistent with PACAP (Ala8Ser/Glu9Asp/Thr11Ile/Ser14Asp/Asp15Ser) comparative CD and fluorescence spectroscopy, NMR, diminished ligand potency and decreased binding and X-ray crystallography analyses of (truncated and affinity by more than 10-fold (Hareter et al., 1997). chimeric forms of) GLP-1 and exendin-4 (Runge et al., Substituting two to three residues in the N-terminal 2007), implying that the differential affinity for the part of GLP-1 by corresponding residues from glucagon ECD can be explained by the higher a-helical propen- (Ala8Ser/Glu9Gln), peptide histidine (Glu9Asp/ sity of exendin-4 in solution and by stronger (ionic) Thr11Ile), or PACAP (Ala8Ser/Glu9Asp/Thr11Ile), in con- interactions between GLP-1R and exendin-4, compared trast, had only small effects on affinity for GLP-1R. with GLP-1 (see above). GLP-1/glucagon chimera ex- 3. Substituted GLP-1 Analogs. In addition to ligand periments showed that a peptide ligand consisting of (and receptor) truncation and chimera studies, several the N-terminal part of glucagon combined with the site-directed substitution experiments were performed C-terminal part of GLP-1 has high affinity for both to provide more detailed information regarding the GCGR and GLP-1R (Hjorth et al., 1994). Substitution of molecular determinants of GLP-1/GLP-1R binding and N-terminal GLP-1 residues with corresponding glu- selectivity. cagon residues [e.g., (Ala8Ser/Glu9Gln)-GLP-1 and a. GLP-1 hydrophobic region I. Point substitution of (Val16Tyr/Ser12Lys)-GLP-1] maintains high GLP-1R– Phe28, Ile29, and Leu32 residues into Ala all had a binding and low GCGR-binding affinities, whereas significant negative impact on GLP-1–binding affinity substitution of N-terminal glucagon residues with and potency, confirming the important role of this corresponding GLP-1 residues decreases GCGR bind- hydrophobic region I in the C terminus of GLP-1 in ing and maintains low GLP-1R–binding affinities. In binding the ECD of GLP-1R (Adelhorst et al., 1994; contrast, substitution of C-terminal GLP-1 residues Gallwitz et al., 1994). The effect of substitution on with corresponding glucagon residues decreases GLP- binding affinity (GLP-1 radioligand competition IC50) 1R binding and maintains low GCGR-binding affini- and potency (cAMP activity EC50) was, however, much ties, whereas replacement of, for example, the last larger for Phe28 (1300/1000-fold decrease in affinity/ three C-terminal residues of glucagon (Met33,Asn34, potency) and Ile29 (93/27-fold reduction) compared with and Thr35) with the corresponding GLP-1 resi- Leu32 (17/twofold decrease), indicating that interac- dues (Val33,Lys34,andGly35) and an additional tions of Phe28 and Ile29 with the conserved hydrophobic C-terminal GLP-1 residue (Arg36) increases GLP-1R core of the ECD of GLP-1R are particularly important binding with affinity for GCGR remaining moderate. (Adelhorst et al., 1994). Substitution of Ala24 by Arg (the Glucagon/GLP-1 chimeras were combined with GCGR/ corresponding residue in glucagon) did not affect GLP-1 GLP-1R chimeras to identify the receptor domains that affinity, whereas Val33Ala (glucagon mimicking) and interact with N-terminal and C-terminal parts of glucagon Ala30Gln only had a moderate 5- to 6-fold 966 de Graaf et al. effect on GLP-1 affinity (Adelhorst et al., 1994). These and Thr13Ala substitutions (133-fold decrease), whereas results imply that apolar interactions of Ala24 and Val33 GLP-1 potency was only significantly affected by the latter with the ECD of GLP-1R [observed in the GLP-1–bound two substitutions. These results suggest that Phe12 and GLP-1R ECD crystal structure (Underwood et al., Thr13 play a more important role in stabilizing the 2010)] are not essential for GLP-1R binding and suggest N-capped conformation via intramolecular interactions that GLP-1 residues at positions 24 (Ala/Arg) and within GLP-1 and/or in stabilizing the activated confor- 30 (Ala/Gln) are not key determinants of GLP-1R/ mation of GLP-1R via intermolecular interactions with GCGR selectivity. the receptor. b. GLP-1 hydrophobic region II. Substitution of Val16, e. N terminus of GLP-1. Substitution studies indi- Tyr19,andLeu20 into Ala decreased GLP-1 affinity by six- cate that the electrostatic, steric, and conformational fold (Val16,Leu20) to 19-fold (Tyr19), and had similar properties of the four residues following the N-capping negative effects on potency (Adelhorst et al., 1994), motif of GLP-1 (His7, Ala8, Glu9, Gly10) are important showing that Tyr19 in particular is an important determinants for GLP-1R binding and/or activation. interaction site in hydrophobic region II located in Substitution of His7 by Phe did not affect binding themiddleoftheGLP-1a-helix. affinity or ligand potency, whereas substitution with c. Polar residues in a-helix. substitution of positively ionizable (Arg, Lys), smaller (Ala), or larger most polar residues in the a-helix of GLP-1 had either (Trp, GIP-mimicking Tyr) residues diminished binding no significant (Ser14, Ser17, Ser18, Gly22, Gln23, Trp32)or affinity (Adelhorst et al., 1994; Hareter et al., 1997; only a moderate 6-fold effect (Lys26, Lys34) on GLP- Gallwitz et al., 2000; Sarrauste de Menthiere et al., 1R–binding affinity and potency, with the exception of 2004). Altogether, these amino acid replacement data the three negatively charged residues Asp15, Glu21, and suggest that a small aromatic residue is required at Glu27 (Adelhorst et al., 1994). Replacement of Asp15 by position 7 that is sterically compatible with the GLP1-R Ala resulted in a 41-fold decrease in affinity and loss of binding site. The Ala8-Val and the exendin-4–mimicking potency (Adelhorst et al., 1994). A recent systematic Ala8-Gly mutants only showed a small (two- to threefold) mutation study combining GLP-1 (Asp15Glu, Asp15Lys, decrease in binding affinity and potency, the Ala8-Ser Asp15Arg) and GLP-1R (residues L379ECL3R/E, analog displayed moderate to large impact (4- to 10-fold) R380ECL3D/G, F381ECL3R/E) demonstrated that an on binding affinity, whereas substitution of Ala8 with ionic interaction between Asp15 and R380ECL3 in the Leu or Thr reduced GLP-1R binding (Adelhorst et al., third extracellular loop of GLP-1R is indeed required for 1994; Hareter et al., 1997; Deacon et al., 1998; Burcelin ligand recognition and receptor activation (Moon et al., et al., 1999). These observations demonstrate that only 2015). The diminished GLP-1 binding of the GLP-1R small residues are tolerated at position 8, suggesting R380ECL3D mutant was partially restored by Asp15Glu that steric constraints of the GLP-1R binding site and/or substitution and almost fully restored by the Asp15Lys conformational flexibility of the N-terminal region and inverted Asp15Arg substitutions of GLP-1. The around Ala8 play a crucial role in GLP-1 binding. Sub- abolished potency of GLP-1 for the GLP-1R R380ECL3D stitution of Glu9 with Asp, Met, or Leu did not signifi- was partially restored by the Asp15Lys and inverted cantly affect ligand affinity/potency; substitution with Asp15Arg substitutions. Replacement of Glu21 by Ala Ser, Tyr, Phe, or Pro moderately decreased affinity (5- to and Gly significantly decreased GLP-1 affinity by 10-fold); whereas replacement with Ala, Lys, and Val 15-fold and 60-fold, respectively (Adelhorst et al., reduced ligand binding (Xiao et al., 2001; Green et al., 1994; Watanabe et al., 1994), indicating that the side 2003, 2004; Sarrauste de Menthiere et al., 2004). The chain of Glu21 plays a key role in GLP-1 binding. substitution data of Glu9 indicate that residues with Glu27Ala substitution had a moderate sixfold effect on similar negative charge (Asp) or comparable size (Met, binding affinity, but a greater, 240-fold, effect on Leu) are preferred, and positively charged moieties (Lys) 10 potency, albeit with a large S.E.M. in the EC50 of the are not tolerated at position 9. Substitution of Gly by mutant (Adelhorst et al., 1994). Replacement of Glu27 by Ala, Asp, Glu, His, Lys, or Arg diminished both GLP-1 Lys did not have a significant effect on binding affinity affinity and potency (Adelhorst et al., 1994; Moon et al., or potency (Watanabe et al., 1994), suggesting that a 2015), demonstrating the essential function of this res- charged/polar residue at position 27 may facilitate GLP- idue in GLP-1R binding. Combined GLP-1 and GLP-1R 1R activation, possibly by stabilizing the a-helix of GLP-1 mutation studies showed that diminished binding affin- and/or stabilizing the active conformation of GLP-1R. ity and potency of GLP-1 for the GLP-1R R380ECL3D d. N-capping motif in GLP-1. Alanine substitution mutant could, for a small part, be recovered by the GLP-1 of Thr11, Phe12, and Thr13, the three residues in GLP-1 Gly10-Arg mutant (Moon et al., 2015), indicating that the that are proposed to stabilize the N-capped conforma- Gly/Arg10 is in the vicinity of R/D380ECL3. tion (Inooka et al., 2001; Neumann et al., 2008) of the N In summary, combining GLP-1 truncation, chimera, terminus of GLP-1, had a significant impact on ligand and point substitution studies presented in this section affinity. The effect of the Thr11Ala substitution was, how- with structural information on GLP-1 conformation and ever, smaller (13-fold decrease) compared with Phe12Ala interactions with GLP-1R and GLP-1R mutation data, a Glucagon-Like Peptide-1 and Its Receptors 967 binding model for the full-length GLP-1–GLP-1R com- the receptors for secretin, , and cal- plex can be proposed (Fig. 2), in which: 1) hydrophobic citonin, and hence enabled it to be classified within what region I in the C-terminal a-helix of GLP-1 consisting of was then a new branch of the GPCR superfamily (family B, Phe28, Ile29, and Leu32 binds the ECD (as observed in class B, or -like) that now includes the GLP-1–bound GLP-1R ECD crystal structure); 2) a 15 members (Segre and Goldring, 1993; Hoare, 2005). second hydrophobic region II in the middle of GLP-1 2. Receptor Expression. Using RNA enzyme protec- (Val16, Tyr19, Leu20) interacts with the region connect- tion techniques, GLP-1R was found widely expressed in ing the ECD and 7TMD (the so-called stalk region in the the human and mouse pancreas, lung, brain, , GCGR crystal structure); 3) the N terminus (His7-Thr13) heart, and kidney, but was not seen in tissues involved of glucagon interacts with the 7TMD in a so-called in glucose , such as the liver, skeletal muscle, N-capped conformation (stabilized by Thr11,Phe12,Thr13). and fat (Dunphy et al., 1998). DNA hybridization exper- In addition to these main determinants of GLP-1/GLP-1R iments showed that the human GLP-1R (hGLP-1R) gene binding, other residues in the C-terminal/a-helical region is localized to chromosome 6p21, and the size is 40 kb, can play an important role in stabilizing and maintaining containing about 14 exons. Both human and rat recep- the amphiphilic character of the a-helix of GLP-1 or by tors contain 463 amino acids, whereas that of the mouse forming additional hydrophobic (Ala24/Ala25 with the has 489 amino acids, displaying a homology with hGLP- ECD) or ionic/polar (Lys26 with the ECD, D15 with 1R of 91% and 84%, respectively. Although the expres- ECL3) interaction sites with GLP-1R. sion levels of GLP-1R vary among different tissues and cell types, Northern hybridization studies demonstrated III. Glucagon-Like Peptide 1 Receptor that its expression in pancreatic islets, heart, and lungs is significantly higher (Dunphy et al., 1998). In addition, A. Discovery GLP-1R was also found in the lateral septum, thalamus, Between 1979 and early 1980, Habener and colleagues and hippocampus of the rat brain, and the cDNA frag- found that 29–amino-acid pancreatic glucagon was the ment cloned from the brain, heart, and pancreas encodes major bioactive peptide released from the pancreatic the same amino acids of the receptor, suggesting its islets (Goodman et al., 1980a; Lund, 2005). The angler- physiologic relevance in the cardiovascular and central fish islet was thus regarded as a rich source containing nervous systems (Satoh et al., 2000). coding sequences for glucagon-related peptides. Hybrid It is known that the expression of GLP-1R in the islets arrest and hybrid selection of mRNAs encoding two of Langerhans is regulated by glucose and dexametha- preproglucagons led to the first identification of a cDNA sone, but not by the PKA-dependent signaling pathway encoding a 29-residue peptide highly homologous to (Abrahamsen et al., 1995). Starvation and refeeding mammalian glucagon (Goodman et al., 1980b; Lund could influence its expression in the rat hypothalamus et al., 1981), which was then used as a hybridization and other sites of the CNS (MacLusky et al., 2000). probe to screen the anglerfish islet cDNA library for Although our knowledge about the transcriptional reg- other cDNAs to derive the entire coding sequence of ulation mechanisms of GLP-1R is still limited, promoter anglerfish preproglucagon (Lund et al., 1982). analysis indicate that 1) transcription factors Sp1 and In 1982, the first mammalian (hamster) pancreatic Sp3 may play important regulatory roles and 2) homol- preproglucagon cDNA was reported (Bell et al., 1983a), ogous desensitization and internalization are closely which resulted in the rapid isolation and sequencing of related to the intracellular sites of 441/442, 444/445, the human glucagon gene (Bell et al., 1983b), followed and 451/452 (Wildhage et al., 1999). by bovine and rat preproglucagon cDNAs (Heinrich 3. Receptor Biosynthesis. About 5–10% of GPCRs con- et al., 1984). These landmark discoveries led to our tain an N-terminal cleavable (Schulein current concepts of GLP-1 as an incretin and a satiety et al., 2012). Most GPCRs mediate the integration of hormone (Ebert and Creutzfeldt, 1987; Kreymann et al., receptor into by a signal anchor se- 1987; Dupre et al., 1991; Mattson et al., 2003). It was quence that is located at the first TMD (Audigier et al., found that truncated forms of GLP-1 are also biologi- 1987; Wallin and von Heijne, 1995). Both types of signal cally active in stimulating both insulin gene transcrip- sequences regulate the endoplasmic reticulum (ER) tion and insulin secretion (Mojsov et al., 1986; Drucker signal process at the beginning of the secretory path- et al., 1987; Fehmann and Habener, 1991; II. Glucagon- way (Belin et al., 1996; Kochl et al., 2002). Like Peptide-1). The signal peptide is a sequence of approximately 1. Receptor Cloning. To better understand the action 20 amino acids in the N terminus of the receptor that of GLP-1, Thorens isolated and characterized the first includes a polar and charged N-terminal (n) region, a cDNA of the rat pancreatic b cell GLP-1 receptor in central hydrophobic (h) region, and a polar C-terminal 1992 (Thorens, 1992), followed by cloning of the human (c) region (Schneider and Fechner, 2004; Clerico et al., receptor in 1993 (Dillon et al., 1993; Graziano et al., 1993; 2008). The C-terminal side regularly contains helix- Thorens et al., 1993), revealing a sequence of 463 resi- breaking proline and residues and small un- dues. This deduced primary sequence resembled that of charged residues at positions 1 and 3 of the cleavage 968 de Graaf et al. site. After synthesis of the receptor’s N-tail in the without the signal peptide sequence was expressed in cytoplasm, the translation ceases by its binding to the human embryonic kidney (HEK) 293 cells and dis- signal recognition particle (SRP) (Halic and Beckmann, played normal functionality with respect to ligand 2005; Shan and Walter, 2005). A complex is binding and cAMP activation, suggesting that the attached to the membranes of ER that includes a GTP- putative signal peptide may not be required for re- dependent interaction between SRP and SRP receptor, ceptor synthesis. Immunoblotting analysis showed translocon gating, protein synthesis, and integration of that the amount of GLP-1R synthesized in HEK293 nascent chains with the bilayer (Kochl et al., 2002). The cells was low without the signal peptide, indicating its signal peptide is finally cleaved off by signal pepti- role in facilitating receptor expression (Ge et al., 2014). dases on the ER membrane. In contrast, signal anchor Epitope tags at the N terminus of GLP-1R were detect- sequences are not cleaved and form a part of the mature able by immunofluorescence and immunoblotting, an protein (Audigier et al., 1987; Wallin and von Heijne, observation that is inconsistent with another report that 1995; Higy et al., 2004; Schulein et al., 2012). In addition studied both signal peptide and the hydrophobic region to recognition and binding of SRP and translocation to after the signal peptide. It was found that the signal ER, the N-terminal signal sequence may play key roles in peptide was cleaved in the mature hGLP-1R, and cell and trafficking. This is highly dependent surface expression was almost abolished by the mutation upon whether the signal sequence is a cleaved signal A21-R that prevented the cleavage, demonstrating that peptide or an uncleaved signal anchor. hydrophobic region after the signal peptide is necessary For GLP-1R, studies on the signal peptides of for efficient hGLP-1R trafficking to the cell surface. corticotropin-releasing factor-1 receptor (CRF1R) and Because glycosylation is vital to cell surface expres- corticotropin-releasing factor-2 receptor give a good sion, cleavage of the signal peptide will affect this model to compare. Both CRF receptors exhibit a high process. In addition, mutating W39ECD,Y69ECD,and probability of N-terminal cleavable signal peptide by Y88ECD of hGLP-1R to alanine eliminated its cell computational prediction. In reality, however, only the surface expression without influencing N-linked gly- signal peptide of CRF1R is cleaved, whereas that of cosylation and cleavage of the signal peptide (Thompson corticotropin-releasing factor-2 receptor is translocated and Kanamarlapudi, 2014). and embedded to the plasma membrane by taking TM1 Clearly, such discrepancies may not only reflect dif- as a signal anchor sequence. It is thus integrated to be a ferences in the methods employed among these three part of the N-terminal ECD: not mediating ER target- studies, but also the cellular background and the com- ing, but contributing to receptor activation (Alken et al., plexity of the underlying mechanism(s). 2005). Interestingly, a mutant of rat CRF1R without the signal peptide sequence was able to translocate to the B. Structure cell surface and display similar biologic function to The human GLP-1R is a 463-residue glycoprotein thewild-type.TheratCRF2aR contains an uncleaved containing an N-terminal signal peptide and various N-terminal signal peptide, and deletion of this resulted glycosylation sites that are essential for the correct in a significant reduction in its membrane expression, trafficking and processing of the receptor (Thorens, suggesting that receptor transportation was affected 1992; Dillon et al., 1993; Graziano et al., 1993; (Rutz et al., 2006). The complexity of these signal Thorens et al., 1993; Goke et al., 1994; Chen et al., sequences highlights the need for experimental verifi- 2010a; Huang et al., 2010). Like all class B GPCRs, cation of the role of the signal sequence in GLP-1R. GLP-1R possesses an N-terminal ECD of 100–150 According to the signal peptide prediction program residues connected to an integral TM domain (7TM) “SignalP 4.0” (Nielsen et al., 1997; Bendtsen et al., that is typical of all GPCRs, having seven a-helices 2004), the sequence of the first 23 amino acids in GLP- (TM1–TM7) separated by ICL1–ICL3 and ECL1–ECL3 1R fits all the criteria of an N-terminal signal peptide. (Palczewski et al., 2000; Siu et al., 2013). However, The existence of a functional signal peptide was dem- despite this structural resemblance to other GPCRs, the onstrated experimentally by Huang et al. (2010), who sequence of the class B 7TMD is devoid of the consensus showed that it was required for GLP-1R synthesis and sequence motifs that typify the class A GPCRs (e.g., was cleaved thereafter. Mutation of the signal peptide and adrenergic receptors). In contrast, the (A21-R) resulted in retention of the receptor within ER, N-terminal ECD of class B GPCRs is a unique domain whereas mutation of E34-R augmented its cell surface found only in this GPCR subfamily and is central to expression when the signal peptide was deleted. The ligand recognition. Class B GPCRs are believed to all amino acid sequence following the signal peptide in the bind their peptide ligands via a common mechanism GLP-1R, G27ECD-W39ECD, is relatively hydrophobic, and known as the two-domain model, in which the ECD first this region may be recognized by SRP (Huang et al., binds to the C-terminal region of the ligand, enabling a 2010). Ge et al. (2014) went deep into the function of this second interaction between the N-terminal region of the signal peptide by use of constructs containing epitope ligand and the 7TMD of the receptor (Bergwitz et al., tags at the N and/or C terminus. A mutant GLP-1R 1996; Hoare, 2005). Glucagon-Like Peptide-1 and Its Receptors 969

1. N-Terminal Domain. potency but did reduce exendin9–39 affinity by sevenfold a. Structure determination of the N-terminal ECD. and Gly2–GLP-1 by 10-fold (Underwood et al., 2010; The two-domain structure of GLP-1R enabled a strategy Patterson et al., 2013). whereby the ECD could be expressed as an isolated b. Site-directed mutagenesis of the ECD. The domain suitable for structural and functional studies N-terminal domain has been the subject of a number that were used to demonstrate its critical role in ligand of mutagenesis studies summarized in Table 1 and Fig. binding (Wilmen et al., 1996; Xiao et al., 2000; Bazarsuren 3 (Wilmen et al., 1997; Tibaduiza et al., 2001; Mann et al., 2002; Al-Sabah and Donnelly, 2003a; López de et al., 2010b; Underwood et al., 2010; Day et al., 2011; Maturana et al., 2003; Mann et al., 2007, 2010b; Runge Koole et al., 2011; Patterson et al., 2013). Residues that et al., 2007, 2008; Underwood et al., 2010). The definitive have been mutated but do not greatly affect GLP-1 study came from X-ray crystallography whereby the affinity/efficacy include P7ECDL, R20ECDK, L32ECDA, isolated ECD of hGLP-1R from an Escherichia coli inclu- T35ECDA, V36ECDA, R44ECDH, E68ECDA, L123ECDA, sion body preparation was incubated with the peptide E127ECDA, E127ECDQ, E128ECDA, E128ECDQ, and ECD antagonist exendin9–39, before being further purified, E128 M. To study a species-selective small-molecule crystallized, and analyzed using X-ray diffraction to yield antagonist, Tibaduiza et al. (2001) mutated W33ECD to a 2.2Å crystal structure (Runge et al., 2008). The protein Ser (the human to rat GLP-1R substitution), as well as to fold closely resembles that of other class B GPCR ECD various other residue types, showing that there was no structures (Parthier et al., 2009) and contained two effect on GLP-1 albeit that the affinity and potency regions of antiparallel b-sheet, three disulfide bonds values were not given for the peptide ligands used. (46ECD–71ECD,62ECD–104ECD,and85ECD–126ECD), Wilmen et al. (1997) substituted six Trp residues in the and an N-terminal a-helix. The core of the structure ECD with Ala and found that, whereas W87A behaved contains six conserved residues (D67ECD,W72ECD, like wild-type GLP-1R in binding and cAMP assays, the P86ECD, R102ECD, G108ECD, and W110ECD), which remaining substitutions at 39, 72, 91, 110, and 120 are critical for the folding stability. Residues on the resulted in the abolition of detectable radioligand bind- a-helix,turn1,loop2,andtheC-terminalregionforma ing. Modest five- to eightfold reductions in GLP-1 po- ligand-binding groove for the antagonist’s well-defined tency have been observed for E68ECDK, P90ECDA, and a-helix (Leu10-Asn28) that interacts with the ECD R121ECDA (Underwood et al., 2010; Day et al., 2011). The using residues within the Glu15ECD-Ser32ECD region, ECD structures suggest that E68ECD is close to the the most critical residues being Val19ECD,Phe22ECD, C-terminal region of the peptide ligand, but, although Ile23ECD,andLeu26ECD, which are deeply buried in it appears to have little significant interaction with the ECD’s groove. The residues on the N-terminal side exendin9–39 in the human receptor, the equivalent 15 ECD of Glu of exendin9–39 do not interact with the ECD, residue in rat GLP-1R, D68 , enhances exendin9–39 although residues 10–14 are nevertheless critical for affinity by forming a hydrogen bond with Ser32 of the high-affinity binding (Runge et al., 2007), presumably peptide (Mann et al., 2010b). Other mutations, Y69ECDA, because they are required to stabilize the helical Y88ECDA, and L89ECDA, have caused catastrophic effects structure of the ligand. The nine-residue C-terminal on the ability of the receptor to be detected in binding or extension of exendin9–39, which has no equivalent in signaling assays (Underwood et al., 2010). GLP-1, plays no significant role in the peptide’s affinity 2. Seven-Transmembrane Domain. at hGLP-1R (Runge et al., 2007; Mann et al., 2010b). A a. Structure of the 7TMD. To date, with the excep- later crystal structure showed that GLP-1 also forms tion of a recently solved electron microscopy map of an a-helix when bound to the ECD, with the principal GCGR (Yang et al., 2015b), there are no experimentally contacts being the equivalent hydrophobic interface determined three-dimensional structures available for formed by residues Ala24,Ala25,Phe28,Ile29,Leu32, any full-length class B GCPR, but, in addition to the and Val33 (Underwood et al., 2010). In contrast to that isolated ECD structures mentioned above, there are in exendin9–39, the ECD-bound a-helix of GLP-1 is now X-ray crystal structures for the 7TMD of human 22 kinked around Gly , as observed in earlier NMR GCGR (Siu et al., 2013; Jazayeri et al., 2016) and CRF1R studies (Thornton and Gorenstein, 1994). The modest (Hollenstein et al., 2013). The partial CRF1R structure eightfold differential affinity between exendin-4 and (PDB: 4K5Y) was solved in the presence of a small- GLP-1 at the isolated membrane-bound ECD of hGLP- molecule antagonist (CP-376395) and contained 12 1R (Mann et al., 2010b) could largely be explained through thermo-stabilizing mutations and a T4 lysozyme fusion ECD E127 , which interacts with exendin9–39 but not GLP-1, partner inserted into ICL2 (Hollenstein et al., 2013). where mutagenesis to Ala resulted in a sevenfold re- The first GCGR 7TMD structure (PDB: 4L6R) was solved ductioninaffinityforexendin9–39 (Underwood et al., 2010; with an N-terminal fusion partner fused at residue Patterson et al., 2013). Leu32ECD also appears to play a 123 (Siu et al., 2013), and it is this structure that is of role in ligand selectivity, although it is less clear from particular interest to understand GLP-1R because the crystal structure how this occurs: substitution of both the receptors and native ligands are closely related. Leu32ECD by Ala had no effect upon GLP-1 affinity or Recently, another GCGR 7TMD crystal structure was 970 de Graaf et al.

TABLE 1 Summary of effects on GLP-1 pharmacology (affinity and ability to activate cAMP pathway) in published site-directed mutagenesis studies of GLP-1R WT (wild-type) refers to mutations that resulted in either ,fivefold or no statistically significant change from wild-type GLP-1R. ND (not determinable) refers to a property that was measured, but for which a value was not determinable. Blank cells mean that the assays used to estimate that particular pharmacological property were not carried out in the cited work. Residues with symbol † refer to data from rat GLP-1R (if different, the equivalent human residue number is displayed in the table to aid comparison). GLP-1 affinity or potency fold-change values with suffix M are from membrane preparations, whereas suffix C is from whole-cell assays. Cell surface expression values below 75% of WT are shown (.75% are shown as WT): a suffix E represents estimation from ELISAs; suffix mic was evaluated from immunofluorescent microscopy; suffix cyt was evaluated by flow cytometry with an anti-Flag antibody; suffix Ag refers to affinity or cell surface expression levels determined from agonist radioligand-binding assays, Ant whereas suffix was from antagonist radioligand-binding assays. DLog tc values relative to WT are shown where .0.5 and were calculated from data where the expression- corrected efficacy term tc had been calculated using the operational model of agonism, as defined in Wootten et al. (2013c). Residues with transmembrane helices are numbered according to Wootten et al. (2013c).

Mutated -Fold Reduction -Fold Reduction Cell Surface Comments and/or Other Residue ‡ Expression Reference to Affinity Potency (% Wild-Type) Observed Effects P7ECD LWTC,Ant WT WTE Koole et al., 2011 R20ECD KWTC,Ant WT WTE Koole et al., 2011 L32ECD AWTM,Ag WT WTAg Underwood et al., 2010 L32ECD A WT Patterson et al., 2013 W33ECD SWTC,Ant Species change (human to Tibaduiza et al., 2001 rat)— the expected lack of effect on GLP-1 pharmacology was implied in text, but no data are shown T35ECD AWT 7%Ag Underwood et al., 2010 Val-36ECD AWTM,Ag WT WTAg Underwood et al., 2010 W39ECD† NDC,Ag Membrane expression Wilmen et al., 1997 confirmed via WB R44ECD HWTC,Ant WT WTE Koole et al., 2011 N63ECD LWTC,Ag WT WTAg Chen et al., 2010a E68ECD AWTM,Ag WT WTAg Underwood et al., 2010 E68ECD K 8 Day et al., 2011 Y69ECD ANDM,Ag ND NDAg Underwood et al., 2010 W72ECD† NDC,Ag Membrane expression Wilmen et al., 1997 confirmed via WB N82ECD LWTC,Ag WT WTAg Chen et al., 2010a W87ECD WTC,Ag WT 24–62% Transient and stable cell Wilmen et al., 1997 lines analyzed Y88ECD ANDM,Ag ND NDAg Underwood et al., 2010 L89ECD ANDM,Ag ND NDAg Underwood et al., 2010 Pro-90ECD AWTM,Ag WT WTAg Underwood et al., 2010 W91ECD† NDC,Ag Membrane expression Wilmen et al., 1997 confirmed via WB W110ECD† NDC,Ag Membrane expression Wilmen et al., 1997 confirmed via WB N115ECD LWTC,Ag WT WTAg Chen et al., 2010a W120ECD† NDC,Ag Membrane expression Wilmen et al., 1997 confirmed via WB R121ECD AWTM,Ag WT WTAg Underwood et al., 2010 L123ECD AWTM,Ag WT 47%Ag Underwood et al., 2010 E127ECD AWTM,Ag WT WTAg Underwood et al., 2010 E127ECD A WT Patterson et al., 2013 E127ECD EWTM,Ag WT WTAg Underwood et al., 2010 E128ECD AWTM,Ag WT WTAg Underwood et al., 2010 E128ECD A WT Patterson et al., 2013 E128ECD QWTM,Ag WT WTAg Underwood et al., 2010 E128ECD M WT Day et al., 2011 R1311.26b NWTC,Ant WT WTE Koole et al., 2011 L1411.36b AWTC,Ag WT 67%cyt Yang et al., 2016 Y1451.40b AWTC,Ag WT 47%cyt Yang et al., 2016 Y1481.43b ANDC,Ag 26 WTcyt Yang et al., 2016 Y1481.43b N15C,Ag 8WTcyt Yang et al., 2016 Y1481.43b FNDC,Ag 14 WTcyt Yang et al., 2016 Y1481.43b F10C,Ant 14 WTcyt Yang et al., 2016 T1491.44b M60C,Ant 14–33 WTE & Ant Beinborn et al., 2005 1.44b C,Ant E T149 M* 250 160 ,50% Emax = ND Koole et al., 2011 For additional residue substiutions, see Koole et al., 2015 T1491.43b MNDC,Ag 59 WTcyt Yang et al., 2016 T1491.43b ANDC,Ag 82 WTcyt Yang et al., 2016 T1491.43b SWTC,Ag WT WTcyt Yang et al., 2016 Y1521.47b A30M,Ant ND 7%Ant Coopman et al., 2011 Y1521.47b HWTC,Ag WT WTcyt Yang et al., 2016 1.50b C,Ant E Ant S155 AWT 10 55% , 48% DLog tc = 0.75 Wootten et al., 2013c G168ICL1 SWTC,Ant WT ,50%E Koole et al., 2011 F169ICL1† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 R170ICL1† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 (continued) Glucagon-Like Peptide-1 and Its Receptors 971

TABLE 1—Continued

Mutated -Fold Reduction -Fold Reduction Cell Surface Comments and/or Other Residue ‡ Expression Reference to Affinity Potency (% Wild-Type) Observed Effects H171ICL1† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 L172ICL1† AWTC,Ag 35%Ag WT cAMP 1027 M GLP-1 Mathi et al., 1997 H173ICL1† AWTC,Ag 48%Ag WT cAMP 1027 M GLP-1 Mathi et al., 1997 C174ICL1† AWTC,Ag 45%Ag 37% cAMP 1027 M GLP-1 Mathi et al., 1997 C174ICL1 A 6 Underwood et al., 2013 C174ICL1 S 7 Underwood et al., 2013 T1752.45b† AWTC,Ag 57%Ag WT cAMP 1027 M GLP-1 Mathi et al., 1997 R1762.46b† AWTC,Ag 13 WTAg 26% cAMP 1027 M GLP-1 Mathi et al., 1997 N1772.47b† AWTC,Ag 22%Ag 43% cAMP 1027 M GLP-1 Mathi et al., 1997 H1802.50b† R21C,Ag WTmic Heller et al., 1996 2.50b C,Ant E Ant H180 AND 12 18% ,ND DLog tc = 0.86 Wootten et al., 2013c N1822.52b† AWTC,Ag 24%Ag “36% of WT” cAMP Xiao et al., 2000 S1862.56b AWTC,Ant WT WTE,Ant Wootten et al., 2013c F1872.57b ANDC,Ag ND 5%cyt Yang et al., 2016 R1902.60b† A .20C,Ag 21%Ag “27% of WT” cAMP Xiao et al., 2000 R1902.60b A32M,Ant 270 7%Ant Coopman et al., 2011 2.60b C,Ant E Ant R190 A20 34 53% ,44 DLog tc = 0.53 Wootten et al., 2013c R1902.60b ANDC,Ag ND WTcyt Yang et al., 2016 R1902.60b KNDC,Ag 17 WTcyt Yang et al., 2016 R1902.60b K32C,Ant 17 WTcyt Yang et al., 2016 L1922.62b S WT Underwood et al., 2011 L1922.62b SWTC,Ag WT WTcyt Yang et al., 2016 V1942.64b AWTC,Ag WT WTcyt Yang et al., 2016 F1952.65b LWT59%Ag Underwood et al., 2011 I1962.66b† SWTC,Ag ND Moon et al., 2012 K1972.67b A28M,Ant 630 57%Ant Coopman et al., 2011 K1972.67b† A5C,Ag 30%Ag “25% of WT” cAMP Xiao et al., 2000 K1972.67b ANDC,Ag ND 51%cyt Yang et al., 2016 K1972.67b INDC,Ag ND 51%cyt Yang et al., 2016 K1972.67b I28C,Ant ND 51%cyt Yang et al., 2016 K1972.67b QNDC,Ag ND WTcyt Yang et al., 2016 K1972.67b RNDC,Ag 23 WTcyt Yang et al., 2016 D1982.68b† A10C,Ag 16%Ag “45% of WT” cAMP Xiao et al., 2000 D1982.68b† A63M,Ant 44 WTAnt López de Maturana and Donnelly, 2002 D1982.68b A43M,Ant 977 66%Ant Coopman et al., 2011 D1982.68b ANDC,Ag ND WTcyt Yang et al., 2016 D1982.68b† NWTM,Ag López de Maturana and Donnelly, 2002 D1982.68b NNDC,Ag 89 WTcyt Yang et al., 2016 D1982.68b ENDC,Ag 434 WTcyt Yang et al., 2016 D1982.68b E58C,Ant 434 WTcyt Yang et al., 2016 A200-L201† V/A WTM,Ag WT López de Maturana et al., 2004 L2012.68b ANDC,Ag ND WTcyt Yang et al., 2016 K202ECL1† AWTC,Ag 45%Ag “71% of WT” cAMP Xiao et al., 2000 K202/W203† A/A WTM,Ag WT López de Maturana et al., 2004 W203ECL1 TWTC,Ag WT WTcyt Yang et al., 2016 M204/Y205† A/A 37M,Ant 51 28%Ant López de Maturana et al., 2004 M204/Y205† V/A 23M,Ag 32 WTAnt López de Maturana et al., 2004 M204/Y205† A/V 29M,Ag 87 WTAnt López de Maturana et al., 2004 M204ECL1† AWTM,Ag WT 44%Ant López de Maturana et al., 2004 M204ECL1 ANDC,Ag 334 WTcyt Yang et al., 2016 M204ECL1 RNDC,Ag 93 WTcyt Yang et al., 2016 Y205ECL1† AWTM,Ag WT WTAnt López de Maturana et al., 2004 Y205ECL1 ANDC,Ag 62 WTcyt Yang et al., 2016 S206/T207† A/A WTM,Ag WT López de Maturana et al., 2004 A208/A209† V/V WTM,Ag WT López de Maturana et al., 2004 Q210Q211† A/A WTM,Ag WT López de Maturana et al., 2004 Q211ECL1 DWTC,Ag WT WTcyt Yang et al., 2016 Q211ECL1 RWTC,Ag WT WTcyt Yang et al., 2016 H212ECL1† AWTC,Ag WTAg “WT cAMP” Xiao et al., 2000 H212ECL1 AWTC,Ag WT WTcyt Yang et al., 2016 H212/Q213† A/A WTM,Ag WT López de Maturana et al., 2004 D215ECL1† AWTC,Ag 51%Ag “57% of WT” cAMP Xiao et al., 2000 W214ECL1 VWTC,Ag WT WTcyt Yang et al., 2016 W214/D215† A/A WTM,Ag WT López de Maturana et al., 2004 G216/L217† A/A WTM,Ag WT López de Maturana et al., 2004 L217ECL1 AWTC,Ag WT WTcyt Yang et al., 2016 L218/S219† A/A WTM,Ag WT López de Maturana et al., 2004 Y220/Q221† A/A WTM,Ag WT López de Maturana et al., 2004 Y220ECL1 DNDC,Ag 105 WTcyt Yang et al., 2016 D2223.25b† AWTC,Ag WTAg “82% of WT” cAMP Xiao et al., 2000 D222/S223† A/A WTM,Ag WT López de Maturana et al., 2004 (continued) 972 de Graaf et al.

TABLE 1—Continued

Mutated -Fold Reduction -Fold Reduction Cell Surface Comments and/or Other Residue ‡ Expression Reference to Affinity Potency (% Wild-Type) Observed Effects L224/G225† A/A WTM,Ag WT López de Maturana et al., 2004 C2263.29b*† A25M,Ant 38 65%Ant Mann et al., 2010a C2263.29b A .90 Underwood et al., 2013 R2273.30b† A .20C,Ag 31%Ag “90% WT” cAMP Xiao et al., 2000 R227/L228† A/A WTM,Ag WT López de Maturana et al., 2004 V229/F230† A/A WTM,Ag WT López de Maturana et al., 2004 L232/M233† V/T 10C,Ag 100 Moon et al., 2012 M2333.36b ANDC,Ag 70 WTcyt Yang et al., 2016 M2333.36b A16C,Ant 70 WTcyt Yang et al., 2016 M2333.36b TNDC,Ag 62 WTcyt Yang et al., 2016 M2333.36b FWTC,Ag WT WTcyt Yang et al., 2016 Q2343.37b A13M,Ant 45 27%Ant Coopman et al., 2011 Q2343.37b ANDC,Ag 151 35%cyt Yang et al., 2016 Q2343.37b NWTC,Ag WT WTcyt Yang et al., 2016 Q2343.37b ENDC,Ag 29 69%cyt Yang et al., 2016 Q2343.37b E17C,Ant 29 69%cyt Yang et al., 2016 Y2353.38b A24M,Ant 23 12%Ant Coopman et al., 2011 C2363.39b A WT Underwood et al., 2013 N2403.43b† A .20C,Ag 14%Ag “8% WT” cAMP Xiao et al., 2000 3.43b C,Ant E,Ant N240 AWT 7WT DLog tc = 0.67 Wootten et al., 2013c N2403.43b QWTC,Ant WT WTE,Ant Wootten et al., 2016 C,Ant E,Ant N240/Q394 A/A WT WT 71% DLog tc = 0.70 Wootten et al., 2016 N240/Q394 Q/N 5C,Ant WT 71%E,Ant Wootten et al., 2016 Y2413.44b AWT42%Ag Underwood et al., 2011 Y2413.44b AWTC,Ag WT WTcyt Yang et al., 2016 E2473.50b AWTC,Ag 64%Ag “39% WT” cAMP Xiao et al., 2000 3.50b C,Ant E Ant E247 AND 14 18% ,ND DLog tc = 0.99 Wootten et al., 2013c F260ICL2 LWTC,Ant WT ,50%E Koole et al., 2011 E262ICL2† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 Q263ICL2† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 R264ICL2† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 I265ICL2† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 F2664.42b† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 K2674.43b† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 L2684.44b† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 L2784.54b M WT Underwood et al., 2011 W2844.60b A32M,Ant 1349 34%Ant Coopman et al., 2011 G2854.61b AWTC,Ant WT WTE Koole et al., 2012a I2864.62b AWTC,Ant WT WTE Koole et al., 2012a V2874.63b AWTC,Ant WT WTE Koole et al., 2012a I286/V287 A/A WT 85% SBM,Ag Dods and Donnelly, 2015 K2884.64b A126C,Ant ND WTE Koole et al., 2012a 4.64b M,Ant Ant K288 A23 5732 WT DLog tc = 1.39 Dods and Donnelly, 2015 K2884.64b† A79M,Ant 251 Al-Sabah and Donnelly, 2003b K2884.64b† L63M,Ant 79 Al-Sabah and Donnelly, 2003b K2884.64b LNDC,Ag ND 70%cyt Yang et al., 2016 K2884.64b† RWTM,Ag WT Al-Sabah and Donnelly, 2003b K288/Y289 A, A 2188 28% SBM,Ag Dods and Donnelly, 2015 Y2894.65b AWTC,Ant WT WTE Koole et al., 2012a Y2894.65b AWTM,Ant WT 20%Ant Dods and Donnelly, 2015 L2904.66b AWTC,Ant WT WTE Koole et al., 2012a Y2914.67b AWTC,Ant WT WTE Koole et al., 2012a L290/Y291† A/A WTM,Ag WT Mann et al., 2010a L290-Y291 A/A WT 99% SBM,Ag Dods and Donnelly, 2015 ECL2 C,Ant E E292 A100 126 WT DLog tc = 0.57 Koole et al., 2012a E292ECL2 ANDC,Ag 33 39%cyt Yang et al., 2016 D293ECL2 A25C,Ant 16 62%E Koole et al., 2012a E292/D293† A/A 8M,Ag 79 Mann et al., 2010a E292/D293 A/A 25 75% SBM,Ag Dods and Donnelly, 2015 ECL2 C,Ant E E294 AWT WT WT DLog tc = 0.65 Koole et al., 2012a G295ECL2 AWTC,Ant WT WTE Koole et al., 2012a E294-G295† A/A WTM,Ag WT Mann et al., 2010a E294-G295 A/A WT 97% SB M,Ag Dods and Donnelly, 2015 C296ECL2*† A18M,Ant WT 23%Ant Mann et al., 2010a C296ECL2 A13C,Ant 126 60E Koole et al., 2012a C296ECL2 SNDC,Ag 96 WTcyt Yang et al., 2016 ECL2 C,Ant E W297 A63 316 WT DLog tc = 1.00 Koole et al., 2012a W297ECL2 ANDC,Ag ND WTcyt Yang et al., 2016 W297ECL2 HNDC,Ag 50 40%cyt Yang et al., 2016 T298ECL2 AWTC,Ant WT WTE Koole et al., 2012a W297/T298† A/A 100M,Ant 50 Mann et al., 2010a; Donnelly, 2012 W297/T298 A/A 22 57% SBM,Ag Dods and Donnelly, 2015 (continued) Glucagon-Like Peptide-1 and Its Receptors 973

TABLE 1—Continued

Mutated -Fold Reduction -Fold Reduction Cell Surface Comments and/or Other Residue ‡ Expression Reference to Affinity Potency (% Wild-Type) Observed Effects R299ECL2 A32C,Ant 85 WTE Koole et al., 2012a ECL2 M,Ant Ant R299 AWT WT 40% DLog tc = 0.60 Dods and Donnelly, 2015 R299ECL2 SNDC,Ag 43 WTcyt Yang et al., 2016 ECL2 C,Ant E N300 A126 501 WT DLog tc = 0.80 Koole et al., 2012a N300ECL2 A36M,Ant 104 18%Ant Dods and Donnelly, 2015 N300ECL2 ANDC,Ag ND 16%cyt Yang et al., 2016 R299/N300† A/A 25M,Ant .3000 Mann et al., 2010a; Donnelly, 2012 R299/N300 A/A 331 41% SBM,Ag Dods and Donnelly, 2015 S301ECL2 AWTC,Ant WT 63%E Koole et al., 2012a ECL2 C,Ant E N302 A25 16 WT DLog tc = 0.53 Koole et al., 2012a S301/N302† A/A WTM,Ag WT Mann et al., 2010a S301/N302 A/A WT 86% SBM,Ag Dods and Donnelly, 2015 N302/M303† V/K WTC,Ag 10 Moon et al., 2012 M303ECL2 AWTC,Ant WT WTE Koole et al., 2012a ECL2 C,Ant E N304 AWT WT 70% DLog tc = 0.74 Koole et al., 2012a N304ECL2 AWTC,Ag WT WTcyt Yang et al., 2016 M303/N304† A/A WTM,Ag WT Mann et al., 2010a M303/N304 A/A WT 91% SBM,Ag Dods and Donnelly, 2015 Y3055.35b A79C,Ant 40 52%E Koole et al., 2012a Y3055.35b AWTM,Ant WT 22%Ant Dods and Donnelly, 2015 W3065.36b ANDC,Ant ND ND No receptor expression Koole et al., 2012a W3065.36 A109M,Ant 206 41%Ant Dods and Donnelly, 2015 W3065.36 ANDC,Ag ND WTcyt Yang et al., 2016 †Y305/W306† A/A 316M,Ant 50 Mann et al., 2010a; Donnelly, 2012 Y305/W306 A/A 263 60% SBM,Ag Dods and Donnelly, 2015 L3075.37b A13C,Ant 25 47%E Koole et al., 2012a L3075.37b A WT 87% SBM,Ag Dods and Donnelly, 2015 I3085.38b A WT 78% SBM,Ag Dods and Donnelly, 2015 L307/I308† A/A 251M,Ant 6 Mann et al., 2010a; Donnelly 2012 I3095.39b AWTM,Ant WT WTAnt Dods and Donnelly, 2015 R3105.40b A10M,Ant 1259 17%Ant Coopman et al., 2011 5.40b M,Ant Ant R310 AWT 1290 19% DLog tc = 0.75 Dods and Donnelly, 2015 I309/R310† A/A 50M,Ant .3000 Mann et al., 2010a; Donnelly, 2012 I309/R310 A/A 13,490 13% SBM,Ag Dods and Donnelly, 2015 L311/P312 A/A WT 91% SBM,Ag Dods and Donnelly, 2015 A3165.46b TWTC,Ant WT ,25%E Koole et al., 2011 5.50b C,Ant E,Ant N320 A18 10 WT DLog tc = 0.50 Wootten et al., 2013c F3215.51b† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 L3225.52b† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 I3235.53b† AWTC,Ag 35%Ant WT cAMP 1027 M GLP-1 Mathi et al., 1997 F3245.54b† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 I325/F326† A/A WTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 V3275.57b† AWTC,Ag 15 WTAg 42% cAMP 1027 M GLP-1 Mathi et al., 1997 I3285.58b† AWTC,Ag 9WTAnt 39% cAMP 1027 M GLP-1 Mathi et al., 1997 C3295.59b† AWTC,Ag WTAnt WT cAMP 1027 M GLP-1 Mathi et al., 1997 I3305.60b AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 V3315.61b† AWTC,Ag 14 WTAg 45% cAMP 1027 M GLP-1 Mathi et al., 1997 I3325.62b† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 A3335.63b† LWTC,Ag WTAg WT cAMP 1027 M GLP-1 Mathi et al., 1997 S3335.63b CWTC,Ant WT ,50%E For additional residue Koole et al., 2011 substiutions, see Koole et al., 2015 K3345.64b† AWTC,Ag WTAg 28% cAMP 1027 M GLP-1 Takhar et al., 1996; Mathi et al., 1997

L3355.65b† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Takhar et al., 1996; Mathi et al., 1997

K3365.66b† LWTC,Ag WTAg WT cAMP 1027 M GLP-1 Takhar et al., 1996; Mathi et al., 1997

K334/K351 Deletions WTC,Ag Ag See paper Takhar et al., 1996 for details C3476.36b A WT Underwood et al., 2013 R3486.37b† G12C,Ag ND WTmic Heller et al., 1996 R3486.37b† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Takhar et al., 1996 L3496.38b† AWTC,Ag 76%Ag WT cAMP 1027 M GLP-1 Takhar et al., 1996 A-3506.39b† ENDC,Ag ,10%Ag 5% cAMP 1027 M GLP-1 Takhar et al., 1996 A-3506.39b† KWTC,Ag 21%Ag 2% cAMP 1027 M GLP-1 Takhar et al., 1996 (continued) 974 de Graaf et al.

TABLE 1—Continued

Mutated -Fold Reduction -Fold Reduction Cell Surface Comments and/or Other Residue ‡ Expression Reference to Affinity Potency (% Wild-Type) Observed Effects K3516.40b† AWTC,Ag WTAg WT cAMP 1027 M GLP-1 Takhar et al., 1996 6.42b C,Ant E Ant T353 AND 22 30% ,ND DLog tc = 0.84 Wootten et al., 2013c H3636.52b A98M,Ant ND 24%Ant Coopman et al., 2011 6.52b C,Ant E Ant H363 A23 459%, 53% DLog tc = 1.71 Wootten et al., 2013c E3646.53b A58M,Ant 15 42%Ant Coopman et al., 2011 6.53b C,Ant E,Ant E364 A25 51% DLog tc = 0.66 Wootten et al., 2016 E3646.53b ANDC,Ag ND 6%cyt Yang et al., 2016 E3646.53b QWTC,Ag WT 42%cyt Yang et al., 2016 E3646.53b Q0.2C,Ant WT 42%cyt Yang et al., 2016 E364/E387 N/Q NDC,Ag ND WTcyt Yang et al., 2016 E3646.53b YWTC,Ag WT WTcyt Yang et al., 2016 E3646.53b DWTC,Ag WT 53%cyt Yang et al., 2016 F3676.56b ANDC,Ag 72 WTcyt Yang et al., 2016 F3676.56b A32C,Ant 72 WTcyt Yang et al., 2016 F3676.56b INDC,Ag 20 WTcyt Yang et al., 2016 F3676.56b H7C,Ag 131 WTcyt Yang et al., 2016 M371ECL3 AWTM,Ant WT 54%Ant Dods and Donnelly, 2015 D372ECL3 AWTM,Ant 59 13%Ant Dods and Donnelly, 2015 E373ECL3 AWTM,Ant WT 28%Ant Dods and Donnelly, 2015 H374ECL3 AWTM,Ant WT 22%Ant Dods and Donnelly, 2015 H374ECL3 AWTC,Ag WT WTcyt Yang et al., 2016 A-375ECL3 A10M,Ant WT WTAnt Dods and Donnelly, 2015 R376ECL3 GWTM,Ant WT WTAnt Dods and Donnelly, 2015 R376ECL3 QWTC,Ag WT WTcyt Yang et al., 2016 G377ECL3 AWTM,Ant WT 19%Ant Dods and Donnelly, 2015 T3787.32b AWTM,Ant WT 12%Ant Dods and Donnelly, 2015 L3797.33b R12C,Ag 141 WTAg Moon et al., 2015 L3797.33b E11C,Ag 165 WTAg Moon et al., 2015 L3797.33b AWTM,Ant WT 31%Ant Dods and Donnelly, 2015 R3807.34b D21C,Ag 1853 WTAg Moon et al., 2015 R3807.34b G4C,Ag 40 WTAg Moon et al., 2015 R3807.34b A128M,Ant 263 68%Ant Dods and Donnelly, 2015 R3807.34b QNDC,Ag ND WTcyt Yang et al., 2016 F3817.35b RWTC,Ag WT WTAg Moon et al., 2015 F3817.35b E .200C,Ag 234 WTAg Moon et al., 2015 F3817.35b AWTM,Ant WT 16%Ant Dods and Donnelly, 2015 F3817.35b SWTC,Ag WT WTcyt Yang et al., 2016 I3827.36b AWTM,Ant WT 23%Ant Dods and Donnelly, 2015 7.37b M,Ant Ant K383 AWT 56 WT DLog tc = 1.18 Dods and Donnelly, 2015 L3847.38b AWTM,Ant WT WTAnt Dods and Donnelly, 2015 L3847.38b ANDC,Ag 41 WTcyt Yang et al., 2016 L3847.38b A48C,Ant 41 WTcyt Yang et al., 2016 L3847.38b VNDC,Ag 16 WTcyt Yang et al., 2016 F3857.40b AWTM,Ant WT WTAnt Dods and Donnelly, 2015 T3867.41b AWTM,Ant WT 18%Ant Dods and Donnelly, 2015 7.42b M,Ant Ant E387 AWT WT 43% DLog tc = 0.52 Dods and Donnelly, 2015 E3877.42b AWTM,Ant WT WTAnt Coopman et al., 2011 E3877.42b D13C,Ag 10 WTcyt Yang et al., 2016 E3877.42b D12C,Ant 10 WTcyt Yang et al., 2016 E3877.42b NWTC,Ag WT WTcyt Yang et al., 2016 L3887.43b ANDC,Ag 208 WTcyt Yang et al., 2016 L3887.43b I5C,Ag 81 WTcyt Yang et al., 2016 L3887.43b FWTC,Ag WT WTcyt Yang et al., 2016 T3917.44b AWTM,Ant WT 64%M,Ant Coopman et al., 2011 T3917.44b AWTWTAg Underwood et al., 2011 S3927.47b AWTC,Ant WT WTE, Ant Wootten et al., 2013c Q3947.49b AWTC,Ant WT WTE, Ant Wootten et al., 2013c Q3947.49b NWTC,Ant WTE,Ant Wootten et al., 2016 M3977.52b LWTC,Ag WT Dong et al., 2012 7.57b C,Ant E Ant Y402 AND 10 21% ,ND DLog tc = 1.59 Wootten et al., 2013c C4037.58b A 5.4 Underwood et al., 2013 N4067.61b AWTC,Ant WT WTE,Ant Wootten et al., 2013c R421CTT QWTC,Ant WT ,50%E Koole et al., 2011 C438 CTT A WT Underwood et al., 2013 C458 CTT A WT Underwood et al., 2013 C462 CTT A WT Underwood et al., 2013

CTT, C-terminal tail; SB, specific binding of radiolabeled ligand; WB, Western blotting. †Note that potency changes can be due to changes in affinity, efficacy, and/or cell surface expression, and hence should be interpreted with caution, especially when either the affinity and/or expression levels are not known. *Also included in double mutations. Glucagon-Like Peptide-1 and Its Receptors 975

Fig. 3. Summary of GLP-1R mutagenesis. A snake plot of GLP-1R from http://www.gpcrdb.org that has been colored (see Key) to highlight the location of signal peptide, glycosylation, and phosphorylation sites, as well as the mutated residues in Table 1. It should be noted that the color coding of 74 of the 195 mutated residues (W39, W72, W87, W91, W110, F169-C174, R176, N177, H180, N182, A200-Q213, W214-G225, R227-F230, L232, M233, E262- L268, F321-I332, K334-K336, and R348-K351) reflects the effects of rat GLP-1R mutations projected on the hGLP-1R amino acid sequence. The color coding of 27 of the 185 residues (K202, W203, S206-Q211, Q213, W214, G216-Q221, S223-G225, R227-F230, L332, M233, I325, and F326) reflects the effect of double mutations, not single-point mutations. Information on the fold change in ligand affinity and potency as well as expression levels of the GLP-1R mutants is reported in Table 1. solved that contained 11 thermo-stabilizing mutations Analogs of MK-0893 bind GLP-1R with moderate micro- and a T4 lysozyme fusion partner inserted into ICL2 molar affinity (Xiong et al., 2012), suggesting that the (PDB: 5EE7) (Jazayeri et al., 2016). In this structure, a GCGR crystal structures may also offer a template for small-molecule antagonist (MK-0893) is bound to an the design of small-molecule ligands that target the same extrahelical allosteric binding site that is distinct from extrahelical allosteric binding site in GLP-1R. Even the orthosteric peptide ligand binding site, and the though the general fold of the TM helices is similar to receptor adopts a similar overall conformation as ob- those observed in classes A and C GPCR crystal struc- served in the previously solved GCGR crystal structure. tures, both CRF1R and GCGR structures assume more 976 de Graaf et al. open conformations toward the extracellular side than Likewise, whereas GLP-1R can signal through several any other GPCR of known structure. In addition to this pathways, this section will focus on the effects of site- open 7TMD pocket, the GCGR structure revealed two directed mutagenesis on signaling through the Gs pro- other distinct structural features that are not observed in tein that raises intracellular cAMP levels. The ability of the CRF1R structure, namely, a long N-terminal exten- GLP-1R to activate other G proteins and pathways will sion of TM helix 1 (defined as stalk region) and a long be examined in more detail below. The mutagenesis helix 8 at the C-terminal end of the receptor that is data are summarized in Table 1 and Fig. 3, with the differently oriented than helix 8 observed in the crystal TM numbering following the convention described by structures of other GPCRs (Hollenstein et al., 2014). Wootten et al. (2013c). Although the distinct orientation of helix 8 in the GCGR i. TM1 (S1291.24b-L1671.62b). The first TM helix of structure may be a result of crystal lattice contacts, the GLP-1R (TM1) contains two single-nucleotide polymor- stalk is proposed to play a role in the positioning of the phism (SNP) sites at positions 1311.26b (Arg/Asn) and ECD relative to the 7TMD (Siu et al., 2013). The electron 1491.44b (Thr/Met). Although the R1311.26bN mutation microscopy map of the full-length GCGR stabilized by did not significantly affect GLP-1 action (Koole et al., 2011), a monoclonal antibody (mAb23) suggests that mAb23 Beinborn and colleagues showed that the T1491.44bM interacts with the ECD (preventing glucagon from mutation (which has been associated with T2DM) led to a binding to the receptor) and stabilizes GCGR in an open 60-fold reduction in GLP-1 affinity and a 30-fold reduction conformation in which the TM helix 1 stalk connects the in potency (Tokuyama et al., 2004; Beinborn et al., 2005). ECD and 7TMD and the ECD is almost perpendicular to The importance of this residue for GLP-1 action has been the membrane surface (Yang et al., 2015b). Hydrogen- confirmed subsequently, demonstrating that the same deuterium exchange studies have indicated that des- mutation resulted in a 250-fold reduced affinity and a 1 9 11 16 His -[Nle -Ala -Ala ]-glucagon-NH2 peptide antagonist 160-fold reduced potency, whereas substitution by a variety binding protects the a-helical conformation of the stalk of other residue types also led to compromised affinity and region of GCGR, consistent with molecular dynamics– signaling (Koole et al., 2015), with the exception of the simulation studies (Yang et al., 2015b). Full-length crys- T1491.44bS mutant, which has similar ligand affinity and tal structure of GLP-1R and other class B GPCRs are potency as wild-type GLP-1R (Yang et al., 2016). Mutation required to show whether the stalk region observed in the of Y1481.43b to Ala, Asn, or Phe diminished GLP-1 affinity GCGR 7TMD crystal structure is indeed a conserved and potency (Yang et al., 2016), while that of Y1521.47b to structural feature among secretin-like receptors. The Ala reduced GLP-1 affinity by 30-fold—potency was com- GCGRstructurelacksthepresenceofapeptideor promised as well, although in this case the Bmax was low small-molecule ligand, but there is clearly a deep and (Coopman et al., 2011), whereas Y1521.47bHdoesnotaffect extensive binding pocket comprised of the TM helices either ligand affinity or potency (Yang et al., 2016). Muta- at the extracellular side of the 7TM bundle. A glucagon- tion of the equivalent residue (Y1491.47b)inGCGRalso bound full-length GCGR model (Siu et al., 2013) and caused low expression levels, whereas the mutation to His models of agonist-docked full-length GLP-1R (Dods and was expressed well and displayed sixfold reduced affinity. Donnelly, 2015; Wootten et al., 2016; Yang et al., 2016) Mutation of S1551.50b to Ala, in contrast, reduced coupling have been proposed based on information from a variety [efficacy (Emax), i.e., the maximal response = 38%, DLog tc = of sources, including the crystal structures of the GCGR 0.75] with a minor effect on affinity (Wootten et al., 2013c). 7TMD (Siu et al., 2013), the antibody-bound GCGR ECD ii. ICL1 (G168-C174). The first residue in the loop is (Koth et al., 2012), the GLP-1–bound GLP-1R ECD the site of a known SNP (Gly to Ser), but the simulated (Underwood et al., 2010), and molecular dynamics sim- mutation had no effect on GLP-1 action (Koole et al., 2011). ulations (Wootten et al., 2016; Yang et al., 2016). Residues comprising the region between F169ICL1 and Although such models can be largely consistent with C174ICL1 in the first intracellular loop were systematically the results of mutation studies of GLP-1R (Table 1) (Dods mutated to Ala, but no significant effect upon GLP-1 action and Donnelly, 2015; Wootten et al., 2016; Yang et al., was found (Mathi et al., 1997). However, C174ICL1 has also 2016), and can provide insights into GLP-1R–specific been mutated and exhibited a sixfold reduced potency mechanisms of ligand-binding selectivity (Yang et al., when mutated to Ala and a sevenfold reduced potency 2016) and biased signaling (Wootten et al., 2016), a when mutated to Ser (Underwood et al., 2013). complete interpretation of the mutagenesis data requires iii. TM2 (T1752.45b-L2012.71b). A total of 17 sites a high-resolution structure of the ligand-bound GLP-1R, within TM2 have been subjected to site-directed muta- and, hence, progress in this area is required and keenly genesis (Mathi et al., 1997; Xiao et al., 2000; López de anticipated. Maturana and Donnelly, 2002; Underwood et al., 2011; b. Mutagenesis of the 7TMD. Although there are Coopman et al., 2011; Moon et al., 2012; Wootten et al., numerous molecular pharmacological analyses of vari- 2013b; Yang et al., 2016). Of these, eight mutations ous ligands (both peptidic and nonpeptidic) acting at displayed GLP-1 pharmacology similar to the wild-type GLP-1R, this section of the review will focus only on receptor (T1752.45b,N1772.47b, N1822.52b, and A2002.70b those studies that highlight the action of GLP-1 itself. in rat GLP-1R, and S1862.56b, L1922.62b, V1942.64b, and Glucagon-Like Peptide-1 and Its Receptors 977

F1952.65b in hGLP-1R). In contrast, R1762.46b displayed M204ECL1-Y205 ECL1 displayed some change from the compromised signaling with approximately 13-fold wild-type (87-fold decrease in potency and 37-fold de- ECL1 ECL1 right-shifted potency and Emax reduced to 26% (Mathi crease in affinity). M204 A and Y205 A single et al., 1997). The mutation of the conserved H1802.50b to mutants exhibited wild-type GLP-1 pharmacology in Arg resulted in a 21-fold reduced affinity (Heller et al., rat GLP-1R (López de Maturana et al., 2004), but dimin- 1996), whereas substitution to Ala had led to no detect- ished GLP-1 affinity and potency in hGLP-1R in another ECL1 able binding, a 12-fold reduction in potency, and an Emax study (Yang et al., 2016). Although the W214 - 2.60 ECL1 of 30% (DLog tc = 0.86) (Wootten et al., 2013c). R190 D215 double mutation to Ala showed wild-type has been mutated in multiple different studies: Xiao properties, Xiao et al. (2000) have reported that the et al. (2000) found that its mutation to Ala in rat GLP- single D215ECL1-A mutation has a fourfold reduced 1R resulted in .20-fold reduced affinity and only 27% of affinity and only 57% of the wild-type cAMP production the wild-type response to 100 nM GLP-1; Coopman et al. when treated with 100 nM GLP-1. Yang et al. (2016) (2011) observed a more than 30-fold reduction in affinity showed that Q211ECL1D, Q211ECL1R, H212ECL1A, and 270-fold reduction in potency; Wootten et al. (2013c) W214ECL1V, and L217ECL1A single amino acid mutants described a 20-fold reduction in GLP-1 affinity with a did not affect GLP-1R pharmacology, whereas W203ECL1A ECL1 34-fold reduction in potency and 56% Emax (DLog tc = and Y220 D diminished GLP-1 affinity and potency. 0.53); and Yang et al. (2016) reported a diminished In contrast, the equivalent region in GCGR plays a crit- GLP-1 affinity and potency for the R1902.60bA mutant, ical role in peptide ligand recognition with 15 residues and a 32-fold reduction in GLP-1 affinity with a 17-fold between L198ECL1 and A220ECL1 affecting peptide re- reduction in potency for the R1902.60bK mutant. The sponse (Roberts et al., 2011; Siu et al., 2013). latter GCGR-mimicking R1902.60bK mutant does not v. TM3 (S2193.22b-A2563.59b). Although the substi- affect binding affinity of the glucagon mimetic GLP-1 tution of C2363.39b appeared to have minimal effects on Glu9Gln point mutant, suggesting that this residue GLP-1 pharmacology, the mutation of the conserved plays a role in GLP-1 versus glucagon selectivity (Yang C2263.29b in two studies led to either no detectable et al., 2016). Moon et al. (2012) mutated I1962.66b to Ser activity or a 25-fold reduction in GLP-1 affinity with and observed normal affinity but no activity. K1972.67b 38-fold lower potency; the latter was thought to be has been mutated to Ala by both Xiao et al. (2000) and the result of the breaking of a disulfide bond and the Coopman et al. (2011); the former (rat GLP-1R) showed creation of a hydrophobic side chain at position 296 a fivefold reduction in affinity but only 25% of the wild- (Mann et al., 2010a; Underwood et al., 2013). The type response to 100 nM GLP-1, whereas the latter equivalent cysteine in GCGR (C2243.39b)wasalso (hGLP-1R) observed a 28-fold reduction in affinity and shown to be important for glucagon potency (Prevost a 630-fold reduction in potency. Yang et al. (2016) et al., 2010; Roberts et al., 2011). The remaining showed that mutation of K1972.67b to Gln and Ile also residues spanning S2193.22b to F2303.33b were included diminished GLP-1 affinity and potency in hGLP-1R, in the double-alanine screening by López de Maturana whereas the K1972.67bR mutant reduced GLP-1 po- et al. (2004), and all were found to display wild-type– tency by 23-fold. The equivalent residues have been like GLP-1 pharmacology, although Xiao et al. (2000) mutated in the closely related receptors GCGR (I1942.67b) reported that the single R2273.30bA mutation exhibited and GIPR (R1902.67b), respectively, with significant a more than 20-fold reduced GLP-1 affinity; the equiv- impact on their pharmacology and, in the case of GCGR, alent mutation in GCGR (R2253.30bA) had no detectable an association with Asp3 of the ligand (Perret et al., 2002; peptide binding (Siu et al., 2013). The double substitu- Runge et al., 2003a; Yaqub et al., 2010; Siu et al., 2013). tion of L2323.35b/M2333.36b with V/T reduced affinity by The neighboring residue D1982.68b has also been mu- 10-fold and potency by 100-fold (Moon et al., 2012), tated by several groups: Xiao et al. (2000) observed a whereas the single substitution of M2333.36b with A and 10-fold reduction in affinity and only 20% of the wild-type T abolished GLP-1 affinity and potency (Yang et al., response to 100 nM GLP-1 at rat GLP-1R (D1982.68bA); 2016). The single-alanine substitutions at Q2343.37b and López de Maturana and Donnelly (2002) demonstrated a Y2353.38b, respectively, caused a 13- and 24-fold reduced more than 60-fold reduction in affinity and a more than affinity with 45- and 23-fold reduced potency (Coopman 40-fold reduction in potency for GLP-1 at rat GLP-1R et al., 2011), whereas the M2333.36bF and Q2343.37bN (D1982.68bA); Coopman et al. (2011) described a more mutants did not affect GLP-1 affinity or potency (Yang than 40-fold reduction in affinity but a more substantial et al., 2016). The Q2343.37bE substitution diminished 980-fold reduction in potency at hGLP-1R (D1982.68bA); GLP-1 affinity, which was restored by the mutation of Yang et al. (2016) showed that in addition to D1982.68bA, Glu9 of GLP-1 into the corresponding Gln residue in D1982.68bN and D1982.68bE mutants also diminished glucagon (Yang et al., 2016). Position 3.37b has also GLP-1 and exendin-4 affinity and GLP-1 potency. been implicated in playing a role in the ligand-binding iv. ECL1 (K202-L218). The ECL1 of rat GLP-1R affinity of GCGR and GIPR (Yaqub et al., 2010; Siu was scanned using double-Ala mutagenesis by López de et al., 2013). Replacement of N2403.43b with Ala in rat Maturana et al. (2004), and only the double mutation of GLP-1R reduced affinity by more than 20-fold and 978 de Graaf et al. severely impeded cAMP production induced by 100 nM and hGLP-1R support the importance of these residues GLP-1 (Xiao et al., 2000), whereas the equivalent in GLP-1 pharmacology (Mann et al., 2010a; Dods and replacement in hGLP-1R had minimal effects on affinity Donnelly, 2015). Substitution by Ala of the equivalent ECL2 ECL2 but reduced Emax of GLP-1 to 78% (DLog tc = 0.67; E290 and N291 in GCGR led to, respectively, Wootten et al., 2013c). Likewise, in GCGR the no detectable radioligand binding and a threefold re- N2383.43bA mutation had no effect on affinity, whereas duction in affinity (Siu et al., 2013). The region encom- in GIPR the N2303.43bA mutant reduced potency by passing W297ECL2-N300ECL2 was identified as essential sevenfold. In contrast, substitution of the conserved in rat GLP-1R and hGLP-1R by double-Ala scanning, E2473.50b to Ala in hGLP-1R reduced potency by 14-fold whereas the residues spanning S301ECL2-N304ECL2 did and Emax to 19% (DLog tc = 0.99; Wootten et al., 2013c), not alter GLP-1 pharmacology (Mann et al., 2010a; but the equivalent mutation in rat GLP-1R had no Donnelly, 2012; Dods and Donnelly, 2015). However, major effect (Xiao et al., 2000). individual substitutions in hGLP-1R by Koole et al. vi. ICL2 (F257-I265). Mutations in the second in- (2012a) demonstrated that, along with E294ECL2, tracellular loop to date have not shown any effects on W297ECL2, and R299ECL2, amino acids N300ECL2, GLP-1 pharmacology with the SNP swap Phe/Leu at N302ECL2,andN304ECL2 were also important (Table 1). 260 (Koole et al., 2011) and an Ala scan of E262ICL2- In GCGR, the equivalent tryptophan (W295ECL2)is I265ICL2 (Mathi et al., 1997) displaying wild-type–like indispensible for affinity (Siu et al., 2013). The double properties. mutation of N302ECL2-M303ECL2 to Val-Lys caused a vii. TM4 (F2664.42b-Y2914.62b). Two sites in TM4 small affinity reduction with a 10-fold decrease in have been highlighted as being key to GLP-1 activity. potency (Moon et al., 2012). Residues that appear to be The first is W2844.60b, which, when mutated to Ala, led unimportant for GLP-1–mediated production of cAMP to a 32-fold reduced affinity and a 1350-fold reduced are G295ECL2,T298ECL2,S301ECL2, and M303ECL2 potency (Coopman et al., 2011). The second important (Mann et al., 2010a; Koole et al., 2012a; Dods and site in this helix is K2884.64b, which, when mutated to Donnelly, 2015), which correlates with available data Ala or Leu in rat GLP-1R, respectively, resulted in a 79- for GCGR (Siu et al., 2013). and 63-fold reduction in affinity with a 251- and 79-fold ix. TM5 (W3065.36b-K3365.66b). Three double-Ala lower potency (Al-Sabah and Donnelly, 2003b). The scan mutations have identified the extracellular end of K2884.64bL mutant also abolished GLP-1 affinity and TM5 of rat GLP-1R as being important for GLP-1 potency in hGLP-1R (Yang et al., 2016). However, the recognition, with substitutions of Y3055.35b/W3065.36b K2884.64b to Ala mutation in hGLP-1R caused a 126-fold (this is the ECL2-TM5 interface), L3075.37b/I3085.38b, reduction in affinity with no detectable cAMP produc- and I3095.39b/R3105.40b with Ala residues displaying tion (Koole et al., 2012a) and substitution of K2864.64b in significantly reduced affinity and potency (Mann et al., GCGR abolished radiolabeled ligand binding (Siu et al., 2010a). The equivalent double substitutions in hGLP- 2013). The following mutations did not affect GLP-1 1R supported a role for Y3055.35b/W3065.36b and pharmacology: F2664.42bA, R2674.43bA, L2684.44bA, I3095.39b/R3105.40b. However, whereas single mutations L2784.54bM, G2854.61bA, I2864.62bA/V2874.63bA, of Y3055.35b, L3075.37b, and I3085.38b to Ala did not I2864.62bA, V2874.63bA, Y2894.65bA, L2904.66bA/ significantly alter GLP-1 pharmacology in one study Y2914.67bA, L2904.66bA, and Y2914.67bA (Mathi et al., (Dods and Donnelly, 2015), the individual substitutions 1997; Mann et al., 2010a; Underwood et al., 2010; Koole of Y3055.35b and L3075.37b with Ala resulted in 79-fold et al., 2012a). decreased affinity with 40-fold reduced potency and viii. ECL2 (E292-Y305). The second extracellular 13-fold reduced affinity with 25-fold lowered potency loop is an important region for peptide ligand recogni- (DLog tc = 0.49), respectively, in a second (Koole et al., tion (Mann et al., 2010a; Donnelly, 2012; Koole et al., 2012a). Although W3065.36b was not expressed in levels 2012a; Moon et al., 2012; Dods and Donnelly, 2015). sufficient for analysis in Chinese hamster ovary (CHO) Aside from the key disulfide-forming C296ECL2, which cells (Koole et al., 2012a), in HEK293 cells its mutation has been shown to be crucial for affinity in rat GLP-1R to Ala resulted in .100-fold reduced affinity and .200- and both affinity and coupling in hGLP-1R (Mann et al., fold reduced potency (Dods and Donnelly, 2015). 2010a; Koole et al., 2012a; Underwood et al., 2011), R3105.40bA resulted in a .1200-fold reduced potency, residues E292ECL2, D293ECL2, E294ECL2, W297ECL2, with little effect upon affinity, implying that it plays a R299ECL2,N300ECL2, N302ECL2, and N304ECL2 all play key role in agonist-induced signaling (Coopman et al., 5.35b a role in GLP-1 action (Y305 is involved too—see 2011; Dods and Donnelly, 2015) (DLog tc = 0.75). The TM5 below). Koole et al. (2012a) demonstrated that the equivalent mutation in GIPR, R3005.40bA, caused a ECL2 ECL2 individual substitutions E292 and D293 in 42-fold reduced affinity and 86% Emax (Yaqub et al., hGLP-1R caused, respectively, $100-fold reduced affin- 2010). This first part of TM5 is also involved in glucagon ity and potency (DLog tc = 0.57) and a 25-fold reduced affinity in GCGR, with several mutations showing some affinity with 16-fold reduced potency, but no change in effect on binding (Siu et al., 2013). N3205.50bA had a efficacy. Double mutations of both sites in rat GLP-1R modest effect on GLP-1 action with an 18-fold reduced Glucagon-Like Peptide-1 and Its Receptors 979

7.36b affinity and a 10-fold decreased potency (DLog tc = 0.50) effect, F381 E reduced potency and affinity by .200- (Wootten et al., 2013c). Takhar et al. (1996) and Mathi fold, and L3797.34bRandL3797.34bE resulted in a more et al. (1997) Ala scanned the C-terminal end of TM5, than 10-fold reduced affinity and in the range of 150-fold demonstrating that V3275.57b, I3285.58b, V3315.61b, and decreased potency. R3807.35bAresultedin128-foldre- K3345.64 had reduced GLP-1 affinity and activity. How- duced affinity, without reduced efficacy (Dods and ever, the mutations of A3165.46T, F3215.51A, L3225.52bA, Donnelly, 2015). The R3807.35bQ substitution resulted I3235.53bA, F3245.54bA, V3255.55bA, R3265.56bA, C3295.59bA, in a more than 15-fold reduction in GLP-1 affinity and I3305.60bA, V3325.62bA, S3335.63bA, S3335.63bC, L3355.65bA, abolished potency (Yang et al., 2016), whereas R3807.35bD and K3365.66bA did not greatly alter GLP-1 pharmacology lowered potency by 1850-fold, but affinity was only re- (Mathi et al., 1997; Koole et al., 2011, 2012a). duced by 21-fold, and R3807.35bG decreased potency by x. ICL3 (A337-T343). ICL3 does not appear to play a 40-fold and affinity by fourfold. K3837.38bAshowedno critical role in GLP-1R pharmacology because Takhar significant change in agonist affinity, but displayed re- et al. (1996) screened the entire ICL3 region via deletion ducedpotencyandefficacy(56-foldreducedpotency,DLog mutagenesis in three- or four-residue sections but found tc = 1.18) (Dods and Donnelly, 2015). Mutation of no effect on GLP-1 action, whereas Underwood et al. Y4027.57bA demonstrated a role for this residue in coupling (2013) found that C341ICL3-A also had no effect (Takhar because substitution to Ala resulted in a large decrease in et al., 1996; Underwood et al., 2013). efficacy (DLog tc = 1.59) (Wootten et al., 2013c), whereas xi. TM6 (D3446.33b-F3696.58b). Takhar’sICL3Ala the equivalent mutation in GIPR, Y3927.57bA, caused a screening also extended into the first eight residues of fivefold reduction in potency (Yaqub et al., 2010). Substi- TM6, but no significant effect on GLP-1 action was shown tutions at E3877.42b, T3917.46b,Q3947.49b, M3977.52b,and (Takhar et al., 1996). Whereas Underwood et al. (2013) C4037.58b toAladidnotaffectGLP-1pharmacology confirmed that C3476.36bA also had no effect, Heller et al. (Coopman et al., 2011; Dong et al., 2012; Underwood et al., (1996) showed that R3486.37bG resulted in a 12-fold reduced 2011; Wootten et al., 2013c). Substitution of L3847.39 (Ala, affinity and inability to produce cAMP. T3536.36bAledtoa Val) and L3887.43 (Ala, Ile) into other aliphatic residues 22-fold reduction in potency (DLog tc = 0.84) (Wootten et al., diminished GLP-1 affinity and potency in a similar way as 2013c). The Ala substitution at E3646.53b caused a 58-fold corresponding mutants of homologous L3827.39b and reduction in affinity with a 15-fold reduced potency L3867.43b residues in GCGR. The L3867.43bFdidnotaffect (Coopman et al., 2011), whereas the individual substitu- GLP-1R affinity or potency, whereas the corresponding tions of E3646.53b with Tyr, Asp, or Gln did not affect GLP-1 L3867.43bF mutant of GCGR abolished glucagon affinity affinity or potency (Yang et al., 2016). Interestingly, the and potency (Siu et al., 2013; Yang et al., 2016). The E3646.53bQ mutation resulted in a 50-fold and a 40-fold E3877.42bN mutant did not affect GLP-1 binding or increase in GLP-1– and exendin-4–binding affinity, re- potency, whereas the E3877.42bD mutant decreased GLP-1 spectively, in exendin9–39 radioligand competition studies affinity and potency by 13-fold and 10-fold, respectively, (Yang et al., 2016). Mutation of H3636.52b to Ala resulted in and the double E3646.53bN/E3877.42bQ mutant completely either a 100-fold reduced affinity with no detectable abolished GLP-1 binding (Yang et al., 2016). The recipro- potency (Coopman et al., 2011) or a 23-fold reduced affinity cal Ser8Ala substitution of glucagon restored binding of 7.42b with poor coupling (17% Emax, DLog tc = 1.71). Mutation of the GLP-1R–mimicking D385 EmutantofGCGR F3676.56b into Ala, Ile, and His decreased GLP-1 affinity (Runge et al., 2003a), indicating that this receptor– and potency by 72-fold, 20-fold, and 131-fold, respectively ligand residue pair plays an important role in ligand (Yang et al., 2016). selectivity between GLP-1R and GCGR. xii. ECL3 (V370-G377). Residues from M371ECL3 to xiv. C terminus (C404-S463). The mutations of G377ECL3 have been mutated to Ala (with A376 to Gly), N406-A, R421-Q, C438-A, C458-A, and C462-A had no showing only a modest effect upon GLP-1 pharmacology major effect on GLP-1 action (Koole et al., 2011; (Dods and Donnelly, 2015). D372ECL3A displayed wild- Underwood et al., 2013; Wootten et al., 2013c). Widmann type affinity but a 60-fold reduced potency, although et al. (1996a) investigated the role of serine residues (four analysis with the operational model suggested no effect serine doublets in the C-terminal tail) in receptor desen- upon efficacy; hence, this is likely to be the result of sitization. Single- and double-Ala mutations of the reduced receptor expression (13% wild-type). A375ECL3G doublets S431/S432, S441/S442, S444/S445, and S451/ resulted in a 10-fold reduced affinity for GLP-1. ECL3 S452 were analyzed to demonstrate their role in receptor appears to play a role in ligand and/or ECD recognition in phosphorylation and phorbol-12-myristate-13-acetate– GCGR (Koth et al., 2012). induced desensitization (Widmann et al., 1996b). xiii. TM7 (Y3787.33b-Y4027.57b). Residues from T3787.33b to E3877.42b have been mutated to Ala, suggesting that C. Receptor Function several residues play no major role in GLP-1 recognition— As a member of class B GPCRs, GLP-1R is highly T3787.32b, L3797.33b, F3817.35b, I3827.36b, F3857.39b,and conserved across species, thus underlining the physio- T3867.40b (Dods and Donnelly, 2015). However, Moon logic importance (Huang et al., 2012). GLP-1R mediates et al. (2015) showed that, although F3817.36bRhadno the actions of GLP-1 via the incretin axis that is the 980 de Graaf et al. functional connection between the intestine and the are important, Gaq/11 does not seem to play a major islets of Langerhans in the pancreas. Stimulation of the role. A summary of known pathways involved in GLP-1R with GLP-1 primarily triggers the insulin pancreatic b cell function is illustrated in Fig. 4. release from islet b cells in a glucose-dependent manner i. Insulin Secretion. b cells undergo glucose-stimulated and suppresses glucagon secretion from islet a cells, in insulin secretion. Glucose enters the b cell through glucose addition to several other effects such as delay of gastric transporter 2 and is converted through glycolysis to emptying and inhibition of appetite (Koole et al., 2013a; pyruvate, which enters the mitochondria for oxidative II. Glucagon-Like Peptide-1). phosphorylation, increasing the cytosolic ATP/ADP ratio 1. Signaling. (MacDonald et al., 2005). The increased ATP closes KATP a. Recombinant cells. The physiologic effects of channels, depolarizing the plasma membrane and increas- GLP-1 are mediated by its interaction with GLP-1R ing calcium influx through L-type voltage-dependent and subsequent activation of its downstream signaling calcium channels, causing release of calcium from in- pathways. GLP-1R is pleiotropically coupled and sig- tracellular stores through calcium-induced calcium release nals through G-protein–dependent and independent (CICR) (MacDonald et al., 2005). Increased cytoplasmic mechanisms. It couples to Gas-elevating cAMP when calcium stimulates exocytosis of the insulin secretory overexpressed in recombinant cell lines, including granules. CHO, HEK cells, Chinese hamster lung (CHL) fibro- GLP-1R signaling enhances this glucose-dependent blasts, and COS cells (Wheeler et al., 1993; Widmann insulin secretion through activation of Gas, upregula- et al., 1994; Montrose-Rafizadeh et al., 1999; Wootten tion of cAMP, and subsequent activation of PKA and et al., 2013b). Increased calcium mobilization has also exchange protein activated by cAMP (Epac). PKA is a been observed in CHO, HEK, and COS, but not CHL holoenzyme composed of both regulatory and catalytic cells (Wheeler et al., 1993; Widmann et al., 1994; subunits; the latter are released upon binding of cAMP Montrose-Rafizadeh et al., 1999; Coopman et al., 2010; to the regulatory subunits, leading to phosphorylation Koole et al., 2010). The mechanism behind this, how- of downstream substrates, whereas Epac2 primarily ever, is controversial. Azidoanilide-GTP cross-linking functions as a guanine nucleotide exchange factor for provided evidence for GLP-1R activation of Gaq/11 in small Ras-like G proteins, thereby regulating their CHO cells, whereas in HEK cells GTPgS binding and activity. PKA phosphorylates the sulfonylurea receptor immunoprecipitation studies showed no activation of (SUR1) subunit of the KATP channels, closing them and Gaq or Gai (Montrose-Rafizadeh et al., 1999; Coopman further depolarizing the membrane (Light et al., 2002). et al., 2010). Furthermore, although Wheeler et al. Epac also inhibits KATP by increasing its sensitivity to observed a rapid increase in inositol trisphosphates, ATP (Kang et al., 2008). The cAMP/PKA pathway, in corresponding with increased calcium levels, and sug- concert with the PI3K/protein kinase C (PKC)z pathway gesting activation in GLP-1R/COS (discussed in more detail below), also inhibits voltage- cells, these intermediates did not seem to be involved gated potassium channels, which in response to de- in other studies with COS and HEK cells (Wheeler et al., polarization, opens and allows K+ efflux, repolarizing 1993; Widmann et al., 1994; Coopman et al., 2010). the cell (MacDonald et al., 2003). This delays repolar- Azidoanilide-GTP cross-linking developed by Montrose- ization, allowing increased calcium influx via voltage- Rafizadeh and colleagues also revealed GLP-1R activation dependent calcium channels (MacDonald et al., 2003). of Gai1,2 but not Gai3 in CHO cells. In this cell line, GLP-1 PKA and Epac1/2 are also involved in CICR from the also activates MAPKs, including ERK1/2 (Montrose- ER, which is mediated by PKA via the inositol 1,4,5- Rafizadeh et al., 1999; Koole et al., 2010), and p38, trisphosphate receptor and by Epac1/2 through ryano- through a cholera toxin–dependent pathway (Montrose- dine receptors, increasing intracellular calcium (Kang Rafizadeh et al., 1999). GLP-1R can also elicit G-protein– et al., 2003; Tsuboi et al., 2003; Dyachok and Gylfe, independent signaling, via recruitment of the regulatory/ 2004). These mechanisms, therefore, enhance the scaffolding b-arrestin proteins (Sonoda et al., 2008; ability of b cells to exocytose insulin secretory granules. Quoyer et al., 2010). b-arrestin 2 recruitment has been There is evidence that CICR also contributes to in- observed in CHO, HEK293, and COS-7 cells, and creased mitochondrial ATP, in conjunction with GLP-1– b-arrestin 1 in CHO cells (Jorgensen et al., 2005; induced increases in ATP (Tsuboi et al., 2003). It was Schelshorn et al., 2012; Wootten et al., 2013b). proposed that the intracellular calcium may enter the b. Pancreatic b cells. Research has largely focused mitochondria to activate mitochondrial dehydrogenases on characterizing the GLP-1R signaling in b cells, to increase ATP production (Tsuboi et al., 2003). PKA where it mediates increased insulin secretion, storage, and Epac also seem to have a role in the exocytosis of and synthesis as well as increased b cell mass. Consis- insulin secretory granules, with PKA phosphorylating tent with the findings in recombinant cells, elevated snapin, a protein that is vital to the regulation of vesicle cAMP is vital in b cells for glucose-dependent insulin assembly, and Rab-3–interacting molecule (Rim) and secretion mediated by GLP-1R, and, although calcium Munc-131, which are involved in vesicle fusion (Kwan mobilization, phosphorylation of ERK, and b-arrestin et al., 2007; Song et al., 2011). Epac2 also interacts with Glucagon-Like Peptide-1 and Its Receptors 981

Fig. 4. Summary of the main characterized pathways of glucose and GLP-1 signaling in the pancreatic b cell. Glucose enters the cell through glucose transporter 2 and undergoes glycolysis to produce pyruvate that enters the mitochondria for oxidative metabolism and ATP production. This increase in cytosolic ATP closes the KATP channels, depolarizing the membrane and opening the voltage-dependent calcium channels, increasing calcium influx into the cell, causing insulin exocytosis. GLP-1 increases insulin exocytosis through a number of mechanisms. GLP-1R couples to Gas, activates adenylate cyclase that converts ATP to cAMP, and mobilizes two downstream effectors, PKA and Epac. These have a range of effects, including closing KATP channels, enhancing fusion of insulin secretory granules with the membrane, whereas PKA also closes Kv channels, inhibiting membrane repolarization. PKA and Epac also increase intracellular calcium by facilitating CICR through the opening of inositol 1,4,5-trisphosphate receptor and ryanodine receptor calcium channels, respectively. This increase in calcium has also been proposed to upregulate mitochondrial ATP production and activate calcineurin, so nuclear factor of activated T cells promotes insulin gene transcription to increase insulin stores. Alongside increasing insulin synthesis and exocytosis, GLP-1 signals through a number of pathways to increase b cell mass. PKA reduces ER stress through ATF-Gadd34 signaling, increases b cell neogenesis by activating cyclin D, and elevates the expression of insulin receptor substrate 2 (IRS2), a b cell survival factor, as well as anti-apoptotic proteins Bcl-2 and Bcl-xL through CREB. PI3K is activated by IRS2 and transactivation of epidermal receptor, and this further promotes increased b cell mass through upregulation of PDX-1 and nuclear factor kB, which upregulates anti-apoptotic Bcl-2 /Bcl-xL and inhibitor of apoptosis protein-2.

Rim2 and Piccolo, a calcium sensor, to form a complex region zipper proteins, similar in structure to that is important for insulin secretion (Fujimoto et al., CREB, which bind to the cAMP-responsive element 2002). Studies in islets of phospholipase C and Epac site to upregulate insulin transcription in a cAMP/ knockout mice also suggest that Epac2 acts via Rap1- PKA-independent mechanism (Skoglund et al., 2000; regulated phospholipase C« to cause calcium-induced Chepurny et al., 2002). The upregulation of the insulin exocytosis (Shibasaki et al., 2007; Dzhura et al., expression and activity of transcription factor pancreatic- 2011). GLP-1 enhances glucokinase activity through duodenum homeobox-1 (PDX-1) and its increased activity Epac2 in a Rim2/Rab3A-dependent manner, sensitizing also promote insulin transcription and biosynthesis b cells to glucose. Knockdown of b-arrestin 1 in cultured via a PKA-mediated mechanism (Wang et al., 1999, pancreatic cells resulted in a reduction of both GLP1- 2001). Glucose and GLP-1 potentiate insulin gene mediated cAMP production and insulin secretion (Sonoda transcription, by increasing calcium levels, activating et al., 2008). Evidence suggests that this occurs via an calcineurin that dephosphorylates nuclear factor of increase in b-arrestin 1–mediated ERK1/2 phosphoryla- activated T cells, resulting in its nuclear localization tion that enhances cAMP response element-binding pro- and the promotion of insulin gene transcription (Lawrence tein (CREB) activation and cAMP levels, subsequently et al., 2002). contributing to insulin secretion (Quoyer et al., 2010). iii. b Cell Survival, Proliferation, and Neogenesis. ii. Insulin Synthesis and Storage. GLP-1 also acts to GLP-1 promotes b cell proliferation, neogenesis, and increase insulin stores in b cells by promoting insulin inhibition of apoptosis in rat models of T2DM (Buteau, gene transcription, its mRNA stability, and biosynthe- 2011). GLP-1R activation facilitates the release of sis. This occurs in both cAMP/PKA-dependent and inde- b-cellulin by membrane-bound metalloproteinases, in- pendent manners (Baggio and Drucker, 2007). Studies ducing transactivation of re- with the rat insulin I promoter have implicated basic ceptor, which then signals through PI3K and activates 982 de Graaf et al.

Akt/protein kinase B (PKB), PKCz, and p38 MAPK and p70s6k that perhaps are involved in other GLP-1 effects ERK downstream (Buteau, 2011). (Redondo et al., 2003). However, in both studies, the PDX-1 is also critical in increasing b cell mass due to presence of GLP-1R in these hepatocytes was not its proliferative and anti-apoptotic effects (Li et al., confirmed. 2005). As well as the increased activity mentioned Signaling mechanisms for fatty acid oxidation and above, GLP-1R enhances its expression. Akt inhibits insulin sensitization, important in reducing hepatic forkhead transcription factor (Foxo1) action through its steatosis, however, have been characterized in rat he- nuclear exclusion, relieving its inhibition on expression patocytes confirmed to express GLP-1R (Svegliati-Baroni of forkhead box protein A2, which controls PDX-1 et al., 2011). Exendin-4 treatment resulted in increased expression (Kitamura et al., 2002; Buteau et al., peroxisome proliferator-activated receptor (PPAR) activ- 2006). In addition, GLP-1–mediated activation of PKA ity and PPARg expression through the activation of PI3K upregulates CREB, increasing the expression of IRS2, a and 59 AMP-activated protein kinase (AMPK) pathways b cell survival factor, leading to activation of PI3K/PKB (Svegliati-Baroni et al., 2011). Increased PPAR activity and the anti-apoptotic protein Bcl-2 (Wilson et al., 1996; induced the transcription of fatty acid b-oxidizing en- Rhodes and White, 2002; Jhala et al., 2003). Bcl-2 and zymes such as acyl-coenzyme A oxidase 1-palmitoyl and Bcl-xL are also upregulated through the activation of carnitine palmitoyltransferase 1 A, thereby reducing fatty nuclear factor kB downstream of PKB (Buteau et al., acid levels in hepatocytes (Svegliati-Baroni et al., 2011). 2004). Nuclear factor kB increases expression of in- Increased PPARg expression allowed increased insulin hibitor of apoptosis protein-2, thereby preventing apo- sensitization through the reduction of Ser307 c-Jun ptosis (Buteau et al., 2004). GLP-1 signaling has also N-terminal kinase phosphorylation (Svegliati-Baroni been implicated in downregulation of proapoptotic et al., 2011). GLP-1R activation also seems to reduce caspase-3 and decrease in the cleavage of poly-ADP- by intersecting the insulin-signaling ribose (Hui et al., 2003; D’Amico et al., 2005). ERK1/2 pathway. In HepG2 and Huh7 cells expressing GLP- signaling via b-arrestin 1 also activates p90RSK, which 1R, exendin-4 treatment led to the phosphorylation of phosphorylates proapoptotic Bad and inactivates it key mediators of the insulin-signaling pathway; PDX- (Quoyer et al., 2010). 1, Akt1, and PKCz and small interfering RNA of GLP- PKA activation of MAPK and cyclin D1 is important in 1R knocked down this phosphorylation for PDX-1 and the transition of the G1/Gs phase essential to cell cycle PKCz (Gupta et al., 2010). progression: they promote b cell neogenesis (Friedrichsen ii. Kidney. GLP-1R activation in the kidney medi- et al., 2006). GLP-1R agonists also improve b cell function ates natriuretic and diuretic effects of GLP-1, including and survival upon ER stress. This is believed to occur decreasing renal proximal tubule reabsorption, improv- through PKA activation of C/EBP homologous protein ing endothelial integrity, and reducing hypertension. and growth arrest and DNA damage-inducible protein The transporter NHE3, which largely mediates this (Gadd34), which prevents the dephosphorylation of reabsorption, is inhibited by GLP-1 through PKA and translation initiator elF2a, allowing the ER to recover Epac. Exendin-4 causes decreased NHE3 function in from stress (Yusta et al., 2006). the porcine kidney epithelial cell line, LLC-PK(1), with c. Extrapancreatic signaling. Although GLP-1 sig- phosphorylation at serine 552, a PKA consensus site. naling mediates many other physiologic effects, and The mechanism by which Epac inhibits the NHE3 GLP-1R is expressed in extrapancreatic tissues, the transporter has not been determined (Carraro-Lacroix mechanistic basis for these effects is less well charac- et al., 2009). It is known that angiotensin II signaling terized. Therefore, there is limited knowledge regarding increases NHE3 activity, and oxidative stress is the underlying signaling mechanisms, although some inhibited by GLP-1R–mediated cAMP elevation. In important pathways and molecules have been identified glomerular endothelial cells, exendin-4 promoted in a limited subset of tissues. PKA-dependent phosphorylation of c-Raf(Ser259), i. Liver. GLP-1 reduces hepatic gluconeogenesis and preventing the activation of the angiotensin II– lipogenesis and increases glycogen formation, but there dependent pathway, p-c-Raf(Ser338)/ERK1/2/plasminogen is some debate over whether these effects are mediated activator inhibitor-1, a pathway upregulated in diabetes by GLP-1R in hepatocytes, or whether the effects may due to a hyperglycemic increase in PKC-b (Mima et al., be indirectly mediated through CNS or insulin release. 2012). GLP-1 promotes glycogen synthesis and decreased GLP-1R may also mediate renal protective effects gluconeogenesis in vitro through upregulation of glyco- through a reduction of PPARa as exendin-4 treatment gen synthase that occurs downstream of PI3K/PKB, decreased PPARa expression in both db/m and db/db PKC, and serine/threonine protein phosphatase 1, and mouse models (Park et al., 2007). This decreased also by reduced expression of gluconeogenetic enzyme expression of PPARa was paralleled by decreased phosphoenol pyruvate carboxykinase in rat hepatocytes transforming growth factor-b1, decreased type IV (Redondo et al., 2003; Raab et al., 2009). In this system, , decreased caspase-3 expression, and re- GLP-1 also increases the phosphorylation of MAPK and duced mesangial expansion (Park et al., 2007). Glucagon-Like Peptide-1 and Its Receptors 983

iii. Adipocytes. GLP-1R signaling has been impli- most robust method to quantify efficacy is the operational cated in increased glucose uptake in human adipocytes, model of Black and Leff (Kenakin and Christopoulos, believed to be mediated by PI3K and MAPK (Sancho 2013), with the transduction ratio of t/KA used to et al., 2007). GLP-1R also promotes increased adipocyte define strength of signaling of an individual ligand for a mass through preadipocyte proliferation and inhibition specific pathway, and this ratio can be determined from of apoptosis, mediated by ERK-, PKC-, and AKT-signaling classic concentration-response experiments (Kenakin pathways (Challa et al., 2012). et al., 2012). iv. Nervous System. GLP-1R expressed in the nu- a. Peptide-mediated signal bias. There are multiple cleus tractus solitarius signals to suppress food intake endogenous peptides that can interact with and activate and reduce body weight, and this is proposed to be me- GLP-1R, including the fully processed GLP-17–36 amide diated through PKA (Hayes et al., 2011). PKA decreases and GLP-17–37 peptides, the extended 1–36 and 1–37 phosphorylation of AMPK and increases phosphorylation forms of these peptides, as well as oxyntomodulin. The of p44/23 MAPKs/mitogen-activated protein kinase search for more stable forms of GLP-1 has also provided a (MEK) (Hayes et al., 2011). range of mimetic peptides, including exendin and mod- v. Cardiovascular System. GLP-R is thought to confer ified forms of GLP-1 that have been developed for cardioprotection through a number of mechanisms, in- therapeutic use (V. Pharmaceutical Development and cluding reducing damage caused by ischemia via inhibit- Therapeutics). Evidence for biased signaling requires ing apoptosis and through oxidative stress, and improving measurement of multiple signaling endpoints. Early energy utilization. GLP-1 is able to reduce infarct size in work characterizing ligands for GLP-1R was largely rat hearts, an effect that is attenuated by inhibition of limited to measurement of cAMP, as the latter is the cAMP, PI3K, and p42/44 MAPK and GLP-1R, and also most well-coupled pathway and critical for the incretin requires activation of the mTOR/p70s6 kinase pathway effect on b cells. Therefore, it is only relatively recently (Bose et al., 2005b, 2007). Murine studies have implicated that evidence for ligand-directed signaling has emerged. additional important signaling molecules. The GLP-1R– Initial studies used heterologously expressed GLP-1R dependent cardioprotective effects of liraglutide in mouse to more broadly examine three canonical pathways that cardiomyocytes were associated with an elevation of have been linked to physiologic signaling in b cells, cAMP and increased caspase-3 activity (Noyan-Ashraf specifically cAMP accumulation, ERK phosphorylation, et al., 2009). In addition, liraglutide-mediated suppression and intracellular calcium (iCa2+) mobilization. Even of glycogen synthase kinase 3-b (GSK-3b)andcaspase-3 with this relatively limited assessment of signaling, activation in murine hearts was not evident in GLP-1R quantitative evidence for peptide-mediated signal bias knockout mice (Noyan-Ashraf et al., 2009), confirming that was observed, with oxyntomodulin exhibiting a relative these effects are receptor-dependent. Stimulation of GLP- bias toward pERK over cAMP and iCa2+ signaling com- 1R also decreases H2O2-induced production of reactive pared with either GLP-1 or exendin (Koole et al., 2010), oxygen species and upregulation of antioxidant enzymes and this supported earlier observations of differences in an Epac-dependent manner (Mangmool et al., 2015). in cAMP signaling and b-arrestin recruitment between GLP-1 may signal via AMPK and Akt to improve oxyntomodulin and GLP-17–36 amide (Jorgensen et al., glucose metabolism after an injury to promote recovery. 2007). Likewise, the extended form of GLP-11–36 amide These were important mediators upon exenatide treat- had a relative bias toward pERK, but a loss of iCa2+ ment of TG9 mice that caused improved cardiac con- signaling. Thus, this work demonstrated GLP-1R was tractility, elevated myocardial GLUT4 expression, and subject to the potential for peptide-mediated biased increased uptake of 2-deoxyglucose (Vyas et al., 2011). signaling, although no significant bias was observed 2. Ligand-Directed Signal Bias. As described above, between amidated and nonamidated forms of GLP-17–36 GLP-1R is pleiotropically coupled to a range of signaling or between GLP-1 and exendin for these pathways effectors, each of which can impact on the physiologic (Koole et al., 2010). More extensive analysis of signaling response elicited by receptor activation. This allows the and regulatory pathways that included recruitment of potential for individual ligands to evoke different patterns arrestins revealed additional tiers of biased signaling of response upon interaction with the receptor in what is with both exendin and oxyntomdulin exhibiting stron- termed ligand-directed biased signaling (Shonberg et al., ger arrestin recruitment relative to GLP-17–36 amide, 2014). Such biased signaling and regulation are thought whereas GLP-11–36 did not recruit arrestins (Wootten to arise through the stabilization of distinct ensembles et al., 2013b) (Fig. 5, left panel), at least in this recombi- of receptor conformations that occur through the varying nant system. The synthetic 11-mer peptide, BMS21, chemical contacts between ligands and the receptor. although much less potent than GLP-17–36 amide,hada Although gross changes in signal bias can be recognized relatively similar profile of activation of the canonical as a reversal of potency or efficacy (Kenakin and Miller, pathways (cAMP, pERK, and iCa2+),butdidnotelicit 2010; Koole et al., 2013b; Shonberg et al., 2014), less recruitment of arrestins (Wootten et al., 2013b). Intrigu- dramatic effects require a quantitative framework to ingly, the principal metabolite of GLP-1, GLP-19–36 amide, identify significant differences in ligand response. The displays a relative preservation of pERK signaling 984 de Graaf et al.

(Li et al., 2012a; Wootten et al., 2012), despite a marked have distinct profiles of signaling is not surprising, as attenuation of cAMP production and ability to induce their chemical diversity means that they interact with insulin secretion (Wootten et al., 2012), indicating that the receptor very differently from peptidic ligands. this peptide is strongly biased toward pERK and may Indeed, BETP and compound 2 interact with the in- be capable of signaling in a physiologically relevant tracellular face of GLP-1R in a manner that involves manner. Additional evidence for peptide-mediated sig- covalent modification of C347 in ICL3/TM6 (Nolte et al., naling bias is seen with a yeast assay of chimeric G 2014). protein activation (Weston et al., 2014), consisting of the 3. Molecular Basis for Signal Bias. There is in- yeast G protein GPA1 substituted by five amino acids creasing evidence demonstrating that the conforma- from the human Ga sequence. In this system, exendin, tional change that drives activation transition involves oxyntomodulin, and glucagon exhibited relative bias reordering of hydrogen bond networks and intrahelical toward the GPA1/GaichimeraovertheGPA1/Gas packing. Such a change promotes reorganization of the chimera when compared with signaling by GLP-17–36, intracellular face of the receptor, thereby enabling whereas liraglutide had a similar profile of signaling to interaction with intracellular effectors (Zhou et al., GLP-1 across these two pathways (Weston et al., 2014). 2000; Curran and Engelman, 2003; Angel et al., 2009; This indicates that biased signaling occurs even at the Illergard et al., 2011). These networks are relatively level of G protein recruitment and is consistent with well conserved within GPCR subfamilies and involve stabilization of different ensembles of receptor confor- conserved polar residues (Venkatakrishnan et al., mations by different peptide ligands. 2013). This is best studied for class A GPCRs, and b. Nonpeptide-mediated bias. There has been con- suggests that there is evolutionary conservation of the siderable interest in the development of nonpeptidic mechanisms underpinning activation transition. Al- ligands for GLP-1R as leads with improved bioavail- though there is limited direct homology in the amino ability (Chen et al., 2007; Knudsen et al., 2007; Willard acid sequence of the TM domains between the major et al., 2012b). Although these ligands are principally receptor families, it is expected that such conserved tested in assays of cAMP formation and insulin secretion, polar residues within class B GPCRs play a similarly a number of these compounds have now been evaluated important structural role and contribute to the mecha- across a more broad range of pathways, enabling assess- nism of ligand-directed biased signaling. This view is ment of the extent to which these compounds exhibit bias supported by experiments in which conserved intra- relative to native GLP-1 signaling. The most extensively membranous polar residues in GLP-1R were mutated to studied are the Eli Lilly compound, 4-(3-benzyloxyphenyl)- alanine. A cluster of residues, including R1902.60b, 2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), N2403.43b, H3636.52b, and Q3947.49b, when mutated, and the Novo Nordisk compound 2 (Coopman et al., was shown to change the signaling profile of the recep- 2010; Koole et al., 2010; Cheong et al., 2012; Willard tor for cAMP, pERK, and iCa2+ in both a residue-specific et al., 2012a; Wootten et al., 2013b), but there are now and ligand-specific manner (Wootten et al., 2013c). some data comparing signaling for Boc5 and the Trans- Although differences were particularly marked between Tech Pharma compound TT15 (Wootten et al., 2013b). GLP-1 and oxyntomodulin, selective differences in the Although these compounds have low potency for cAMP effect of mutation were also seen between GLP-1 and production, relative to native GLP-17–36 amide, they also exendin, providing molecular evidence that the mecha- exhibit distinct patterns of signal bias (Koole et al., nism of receptor activation differs between these two 2010; Wootten et al., 2013b). This can be illustrated in a peptides, despite relatively similar overall efficacy for web of bias (Fig. 5, right panel), which reveals that Boc5 the peptides across these pathways at the wild-type and TT15 have similar patterns of signaling via GLP-1R receptor (Wootten et al., 2013c). In addition to these for canonical pathways (cAMP, pERK, and iCa2+), but residues that displayed ligand-specific effects, there have diminished ability to recruit arrestin proteins. In was a series of serine residues (S1551.50b, S1862.56b, contrast, compound 2 and BETP have a relatively and S3927.47b) that played a role in the control of enriched ability to recruit arrestins, but distinct effects receptor-dependent signal bias; however, these had on pERK and iCa2+ mobilization, where BETP trends global effects on all peptides (Wootten et al., 2013c). towards reduced pERK but higher iCa2+, and compound These residues sit at the interface between either TM1 2 has the opposite profile (compared to the reference and TM7 (S1551.50b and S3927.47b) or TM2 and TM3 cAMP pathway and the signaling profile of GLP-1). (S1862.56b) and are most likely involved in tight Additional differences in the behavior of compound packing of these helices. With the solution of crystal 2 and/or BETP have also been noted by others structures of the TM domain of the related CRF1 and (Coopman et al., 2010; Cheong et al., 2012; Thompson glucagon receptors (Hollenstein et al., 2013; Siu et al., and Kanamarlapudi, 2015), albeit that a lack of a 2013; Jazayeri et al., 2016), it is now possible to model quantitative framework for these analyses meant that the location of these residues with improved precision. potential implications for signaling bias were not fully Intriguingly, such modeling indicates that these resi- explored. The fact that these nonpeptidic compounds dues reside at a fulcrum position of the receptor TM Glucagon-Like Peptide-1 and Its Receptors 985

Fig. 5. Web of bias illustrating distinctions in the pattern of signaling of different peptide agonists (left panel) or nonpeptidic modulators (right panel) at GLP-1R. The web of bias plots DDt/KA values on a logarithmic scale for each ligand and for every signaling pathway tested. Formation of these values included normalization to the reference ligand GLP-17–36 amide and the reference pathway, cAMP accumulation. The plots do not provide information on absolute potency, but on relative efficacy for signaling of individual pathways in comparison with that for cAMP. Data are from Koole et al., 2010; Willard et al., 2012a; and Wootten et al., 2013b. bundle, where the splayed helices of the open extra- et al., 2006, 2010, 2012; Gao et al., 2009; Schelshorn cellular face of the receptor converge (Fig. 6), with the et al., 2012). Intriguingly, disruption of the TM4 dimer residues that contribute to ligand-dependent signaling interface of GLP-1R differentially impacted the signal- forming a central interaction network and the smaller ing elicited by agonist peptides with a greater abroga- polar residues that are globally important for signaling tion of iCa2+ mobilization relative to that of either cAMP external to this core. accumulation or ERK phosphorylation (Harikumar et al., Although not broadly conserved across the class B 2012). This indicates that differential ligand-mediated subfamily, there is an additional polar threonine in TM1, signaling could also involve the allosteric interaction of at position 1491.44b, that is the site of a naturally protomers within a receptor dimer. occurring polymorphism leading to incorporation of a In addition to an emerging appreciation of how ligands in this position (Beinborn et al., 2005). can selectively alter key hydrogen-bonding networks, Methionine at this position leads to a marked loss in there is some limited information on how extracellular coupling of the receptor to cAMP accumulation (Fortin loop residues contribute to peptide-mediated bias, in et al., 2010; Koole et al., 2011) and iCa2+ mobilization, particular for ECL2 (Koole et al., 2012a,b). K288ECL2, whereas ERK phosphorylation is relatively preserved C296ECL2, W297ECL2,andN300ECL2 had critical roles in (Koole et al., 2011). This is a global effect for peptide governing signal bias of the receptor, but were also activators of the receptor (Koole et al., 2011). Exploration globally important for peptide signaling. Nonetheless, of the tolerance of this position to different amino acids peptide-specific effects on relative efficacy and signal bias revealed that serine substitution had relatively minimal were most frequently observed for residues 301–305, detrimental effect, suggesting that maintenance of the although R299ECL2A mutation also exhibited different polar nature of this residue is important. However, effects for individual peptides. M303ECL2 appeared to amino acids of similar size, although leading to attenu- play a greater role for exendin and oxyntomodulin actions ation of response in a pathway-specific manner, were the than those of GLP-1 peptides. Interestingly, ECL2 mu- least affected, indicating that side chain packing was also tation was generally more detrimental to exendin- 2+ important (Koole et al., 2015). Thus, this mutation mediated iCa mobilization than GLP-17–36 amide, changes the physiologic bias of the receptor in addition providing additional support for subtle variances in to markedly attenuating peptide-mediated signaling. receptor activation by these two peptides. Although these experiments have been principally 4. Allosteric Modulation. As described above (II. interpreted in the context of in cis signaling of receptor Glucagon-Like Peptide-1 and III. Glucagon-Like Peptide 1 to effectors, GLP-1R, like most, if not all class B GPCRs, Receptor), peptide ligands engage GLP-1R via a diffuse undergoes functionally important dimerization (Harikumar pharmacophore that includes key interactions for affinity 986 de Graaf et al.

Fig. 6. Homology model of GLP-1R illustrating the relative position of key residues involved in the receptor-signaling bias. The modeling indicates that these residues reside at a fulcrum position of the receptor transmembrane bundle, where the splayed helices of the open extracellular face of the receptor converge, with the residues that contribute to ligand-dependent signaling forming a central interaction network (space fill, red) and the smaller polar residues that are globally important for signaling external to this core (space fill, blue). The receptor is displayed in three views at different horizontal rotation. Transmembrane helices are numbered with Roman numerals.

with the N-terminal extracellular domain, as well as 2 has only limited effect on GLP-17–36 amide–induced poorly defined interactions with the extracellular loops cAMP production, it yields a ;30-fold augmentation of and TM domain core that drive receptor activation. oxyntomodulin signaling via this pathway (Koole et al., Nonpeptidic ligands have a distinct mode of binding, 2010; Willard et al., 2012b; Wootten et al., 2013b). Probe and in many cases can do so via topographically distinct, dependence is also seen for effects on the extended GLP-1 allosteric sites from those of the native peptides. Such peptides and exendin, where very limited augmentation ligands can bind simultaneously with peptide ligands of cAMP signaling is observed. A similar pattern of effect and interact in a cooperative manner to alter the binding is observed for BETP in that this compound also aug- and/or efficacy of peptide signaling (and vice versa) ments oxyntomodulin signaling, but has minimal effect (Leach et al., 2007; May et al., 2007; Keov et al., 2011; on GLP-17–36 amide–, exendin-, or GLP-11–36 amide– Wootten et al., 2013a; Gentry et al., 2015). Allosteric mediated cAMP production (Willard et al., 2012b; modulation of GPCRs is now a well-established para- Wootten et al., 2013b). A weak potentiation of compound digm that provides both advantages and challenges for 2–mediated cAMP production (or surrogate readouts drug discovery when compared with classic orthosteric such as cAMP response element–driven luciferase re- ligands (Wootten et al., 2013a). The first characterized porter) has also been reported for interaction of this ligand allosteric modulators of GLP-1R were identified by Novo with truncated forms of exendin, including exendin5–39, Nordisk, and are exemplified by compound 2, which, as exendin7–39, and exendin9–39, even in the absence of any noted above, is an allosteric agonist of the receptor. In measurable intrinsic activity (Coopman et al., 2010; addition to its intrinsic efficacy, this series of compounds Cheong et al., 2012). augmented the binding of radiolabeled GLP-1, indicating Intriguingly, if the cooperative effect of BETP or that it could act as a positive allosteric modulator compound 2 is studied over a broader array of signaling (Knudsen et al., 2007). Nonetheless, there was only endpoints, then marked differences in effect are observed limited impact on the efficacy of GLP-1R for cAMP (Koole et al., 2012a; Li et al., 2012a; Wootten et al., 2012, signaling. A hallmark of allosteric interactions is the 2013b). In contrast to the selective augmentation of phenomenon of probe dependence that describes the cAMP response for oxyntomodulin, the allosteric ligands capacity for different effects, depending upon the ortho- confer a similar, modest enhancement of arrestin re- steric and allosteric ligand combination. This is also cruitment for both GLP-1 and oxyntomodulin (Willard observed for ligands of GLP-1R. Although compound et al., 2012b; Wootten et al., 2013b), although a greater Glucagon-Like Peptide-1 and Its Receptors 987 degree of negative cooperativity is seen on pERK re- exendin potency. At this mutant, compound 2 could re- sponses of GLP-17–36 amide versus oxyntomodulin. Thus, store the potency of both GLP-17–36 amide and exendin the allosteric ligands also alter the signaling bias to that of the wild-type receptor (Koole et al., 2011). mediated by endogenous peptides. This is perhaps not Thus, allosteric modulators have the capacity to alle- surprising as both signaling bias and the cooperative viategeneticdiseasearisingfromthelossoffunction effects of cobound ligands are driven through changes to of GLP-1R. the ensemble of conformations that the receptor samples Disruption of the TM4 dimer interface of GLP-1R also (Wootten et al., 2013a). Indeed, the allosteric ligand- abolishes the intrinsic efficacy of BETP and compound bound receptor can effectively be considered a distinct 2, but the cooperative augmentation of cAMP signaling receptor for the interaction of the natural ligand(s). by oxyntomodulin is maintained, indicating that the Remarkably, both BETP and compound 2 very strik- allosteric effect occurs in cis at least for this class of ingly enhanced the cAMP response of the principal ligands (Harikumar et al., 2012). GLP-1 metabolite, GLP-19–36 amide,byupto;400-fold Although compounds such as BETP and compound in the case of compound 2 (Li et al., 2012a; Wootten 2 were among the first recognized and the most widely et al., 2012). Parallel augmentation of insulin secretion studied allosteric modulators of GLP-1R, a number of was observed in isolated rat islets and in vivo when additional compounds have been reported to act as pharmacological levels of the metabolite were coadmi- modulators of peptide response. This includes the nistered with subthreshold levels of BETP (Wootten flavonol, , which lacks intrinsic activity, but et al., 2012). The augmentation of signaling was prin- could selectively augment calcium signaling of effica- cipally limited to cAMP production, although weak cious peptides such as GLP-17–36 amide, GLP-17–37, and potentiation of pERK and iCa2+ signaling was seen in exendin, albeit that an inhibition of response was seen HEK293 cells recombinantly expressing the receptor with high concentrations of quercetin (Koole et al., 2010; and INS-1E cells (Li et al., 2012a), but not in CHO cells Wootten et al., 2011). This augmentation of iCa2+ was recombinantly expressing the receptor (Wootten et al., dependent upon the presence of a 3-hydroxyl group on 2012), providing further evidence that allosteric modu- the flavone backbone and was improved when a 3949- lators can alter the signal bias of the receptor. None- dihydroxyl modification was present (Wootten et al., theless, the lack of effect on the extended GLP-1 peptide 2011). An additional series of compounds that could indicates that the magnitude of the cooperative effect selectively enhance iCa2+ signaling was recently report- of BETP and compound 2 is not driven solely by the ed following high-throughput screening (Morris et al., intrinsic efficacy of the activating peptide. 2014). Unlike the flavonols, this series of compounds Ligands such as BETP and compound 2 are highly had significant intrinsic efficacy for mobilization of electrophilic and can form adducts with free cysteine iCa2+. An exemplar from this series, termed (S)-9b, was residues (Eng et al., 2013). This is a feature of the action examined in further detail, in which it was reported to of these molecules to form, in particular, the covalent also augment liraglutide-mediated GLP-1R internali- interaction with C3476.36b in ICL3/TM6 that is critical zation in recombinant cells and exendin-mediated in- for both the intrinsic efficacy and the cooperative allo- sulin secretion in primary mouse islets (Morris et al., steric effect (Nolte et al., 2014). Nevertheless, the 2014). This latter effect occurred in both high and low covalent modification of the cysteine is insufficient to glucose, which would be problematic from a therapeutic explain the full extent of activity of these compounds as standpoint, but may also require further investigation to BETP and compound 2 have differences in their profile determine whether this is mediated by GLP-1R. Like- of intrinsic efficacy (see above) and also in their co- wise, compound (S)-9b reduced haloperidol-induced cat- operative effect (Wootten et al., 2013b). Interestingly, alepsy in rats (Morris et al., 2014), but, again, it needs to there is a naturally occurring polymorphism that can be confirmed whether this effect is mediated via GLP-1R. appear at position 333 of the human receptor, being In other work, using virtual screening against a molec- either a serine or a cysteine with the latter occurring ular model of GCGR, de Graaf et al. (2011) identified a only rarely (Koole et al., 2011, 2015). However, the number of compounds that had activity at both GCGR C333ICL3 variant selectively abrogates compound 2– and GLP-1R, including weak antagonists and one com- mediated cAMP production and cooperativity between pound that acted as an inhibitor of glucagon-mediated compound 2 and oxyntomodulin for this pathway (Koole cAMP production at GCGR, but as a weak positive et al., 2011) while maintaining peptide-mediated sig- modulator of GLP-1R–mediated cAMP response. naling, suggesting that the environment surrounding At this point in time, the physiologic or therapeutic C3476.36b is important for the action of compound 2. In implications of biased signaling are largely unknown. contrast to the effect of the C333ICL3 polymorphism, the Despite differences in the signaling/regulatory profile of M1491.44b polymorphic variant has no effect on the in- compounds such as Boc5 or TT15, these compounds (or trinsic efficacy of compound 2, but, as noted above, causes similar analogs) have demonstrated efficacy in modu- a very marked attenuation of peptide-mediated cAMP lation of ex vivo and in vivo insulin secretion (Chen signaling with over 100-fold loss of GLP-17–36 amide or et al., 2007; He et al., 2012). Allosteric compounds such 988 de Graaf et al. as BETP or compound 2 likewise can augment insulin et al., 1996a,b). Homologous desensitization refers to secretion, albeit that this is complicated by endogenous the loss of response to subsequent agonist stimulation circulating peptides. Pharmacologically, both BETP following direct stimulation of the receptor and can occur and compound 2 can augment responses of oxyntomo- through controlling the number of receptors present at dulin and the GLP-1 metabolite (GLP-19–36 amide), and the cell surface or by regulating the efficacy of the this is linked to increased in vivo insulin secretion, at receptors at the cell surface. Heterologous desensitiza- least in the context of threshold doses of the allosteric tion describes receptor activation-independent regula- and orthosteric peptides (Willard et al., 2012b; Wootten tion of receptors as well as mechanisms that occur after et al., 2012). In vitro assessment of the effect of the receptor activation that do not discriminate between modulators on the signaling profile of oxyntomodulin or activated, and nonactivated, receptors (Ferguson, 2001). GLP-19–36 amide indicates that their most prominent Acute incubation of islet cells with native GLP-1 in- effect is to augment cAMP production, suggesting that duces rapid homologous GLP-1R desensitization in vitro this alone may be sufficient to improve insulin secre- (Fehmann and Habener, 1991; Gromada et al., 1996). In tion, at least in the context of the baseline signaling of addition, exendin-4 also produces GLP-1R desensitization the peptides/modulators. The significance for GLP-1R in islet cells following both acute and chronic exposure; function outside of insulin secretion is even less clear, as however, this ligand is associated with a greater degree of there has been very limited assessment of these other desensitization compared with comparable incubations functions and none controlled for the influence of biased with GLP-1 (Baggio et al., 2004). Heterologous desensiti- signaling. A major current limitation is the range of zation has been demonstrated in islets in response to tools available to probe the significance of signaling multiple stimuli. For instance, GLP-1R heterologous de- differences. Most nonpeptidic compounds have low sensitization was observed via acute exposure to phorbol potency, nonfavorable pharmacokinetic profiles, or un- myristate acetate (PMA), an activator of PKC (Widmann known interaction with other targets that limits their et al., 1996a,b; Baggio et al., 2004). In addition, prolonged utility. Exendin is biased relative to GLP-17–36 amide, but hyperglycemia can induce desensitization, followed by this difference is subtle in the context of canonical downregulation with diminished responses to GLP-1R signaling, making interpretation of physiologic data agonists that include weakened insulin secretion, reduced difficult. Oxyntomodulin is the most biased of the char- phosphorylation of CREB, impaired cAMP responses and acterized peptides, displays good affinity for GLP-1R, PKA activity, and a reduction in GLP-1R mRNA (Rajan and has distinctions in its physiologic actions from GLP-1, et al., 2015). but dissecting the importance of this bias is complicated There is also the possibility that heterologous desensi- by its significant interaction with GCGR (Pocai, 2013). tization may occur due to activation of other receptors Nonetheless, this may be a useful tool to study biased expressed in b cells that are involved in tightly controlling signaling in GCGR knockout animals. Additional ap- insulin secretion. To date, this has only been assessed proaches to infer the potential role of selective activation with the ligand for GIPR. GLP-1R can heterodimerize of signaling/regulatory pathways include the generation with GIPR in vitro, with evidence that this heterodime- of knock-in mice that have receptors designed to selec- rization can alter cellular signaling and trafficking pro- tively abrogate signaling via a specific pathway (e.g., G files of GLP-1R (Schelshorn et al., 2012; Roed et al., 2015). protein versus arrestin), although the development of However, despite their overlapping functions for signal- potent biased peptide ligands could also provide novel ing and insulin secretion, GIP does not produce meaning- scope for better understanding this phenomenon. ful heterologous desensitization of GLP-1R in islet cell studies (Rajan et al., 2015). D. Receptor Regulation 2. Underlying Mechanisms. Desensitization is a con- 1. Receptor Desensitization. The activity of GLP-1R, sequence of a number of different mechanisms. They like all GPCRs, is regulated by a coordinated balance include uncoupling the receptor from heterotrimeric G between molecular mechanisms governing receptor proteins in response to receptor phosphorylation, the signaling, desensitization, and resensitization. Recep- internalization of receptors to intramembranous com- tor desensitization, or the reduced responsiveness of partments, and the downregulation of the total com- GPCR signaling to an agonist with time, is an important plement of receptors (Ferguson, 2001). physiologic feedback mechanism that protects against a. GLP-1R phosphorylation. The most rapid mecha- both acute and chronic receptor overstimulation (Ferguson, nism by which GPCRs are desensitized is through the 2001). This has important biologic and therapeutic covalent modification of the receptor as a consequence of implications. phosphorylation by intracellular kinases, either G protein– GLP-1R undergoes rapid and reversible homologous coupled receptor kinases (GRKs) or second messenger and heterologous desensitization that has been ob- kinases (Ferguson, 2001). Both homologous and heterolo- served in recombinant cell systems, in primary islets gous desensitization of GLP-1R are accompanied by of Langerhans, and in insulinoma b cell lines (Fehmann phosphorylation of serine residues within the last 33 amino and Habener, 1991; Gromada et al., 1996; Widmann acids of the C-terminal tail (Widmann et al., 1996a,b). Glucagon-Like Peptide-1 and Its Receptors 989

b. GRKs. For most GPCRs, homologous desensiti- surface in pancreatic islets in conditions of chronic zation is thought to involve phosphorylation by GRKs hyperglycemia (Rajan et al., 2015). In MIN6 cells, reduc- that results in recruitment of b-arrestins. In vitro tions in numbers in conditions of studies in fibroblast cells expressing GLP-1R revealed high glucose were mimicked by overexpression of a that homologous desensitization was associated with constitutively active PKA or continuous activation by phosphorylation of serine residues 441/442, 444/445, forskolin. In addition, inhibition of PKA activity attenu- and 451/452 in the C terminus (Widmann et al., 1997). ated glucose-mediated downregulation of GLP-1R from Desensitization of GLP-1R–mediated cAMP responses the cell surface of these cells. Phosphorylation at serine after a first initial exposure of cells to GLP-1 was strictly 301, within the third intracellular loop of the receptor, is dependent on the extent of phosphorylation with no, implicated in this PKA-mediated activity, with mutation intermediate, or maximum phosphorylation observed in of this residue abolishing glucose-dependent loss of the the presence of one, two, or three of the serine doublet receptor from the cell surface. This was associated with a phosphorylation sites, respectively. loss of an interaction between the receptor and the small Although various groups, using a number of tech- -related modifier, SUMO (Rajan et al., 2015). niques, have demonstrated that stimulation of GLP-1R Sumoylation of GLP-1R causes intracellular retention of leads to recruitment of both GRKs and b-arrestins the receptor and desensitization of receptor signaling as (Jorgensen et al., 2005, 2011; Wootten et al., 2013a), well as prevents resensitization of the receptor back to to date, the direct involvement of these proteins in the cell surface (Rajan et al., 2012). This heterologous desensitization of GLP-1R response has not been dem- desensitization and subsequent downregulation of onstrated. In recombinant systems, GRK2 can interact GLP-1R on the b cell surface by chronic hyperglycemic with GLP-1R in response to stimulation by GLP-1 conditions have substantial implications for the efficacy (Jorgensen et al., 2007, 2011). In addition, GRK2 has of GLP-1–based therapies. Because SUMO expression is been linked to potentiation of b-arrestin 2 recruitment increased in mouse islets exposed to high glucose (Rajan to the receptor. b-arrestin 1 is also recruited to GLP-1R et al., 2012), its interaction with GLP-1R may in part upon activation by agonist ligands (Sonoda et al., 2008; contribute to the reduced efficacy of incretin therapies in Wootten et al., 2013a); however, whether recruitment of some T2DM patients with poorly controlled hyperglyce- these proteins is required for internalization is cur- mia (Fritsche et al., 2000; Stumvoll et al., 2002; Kjems rently not clear. et al., 2003). c. Second messenger protein kinases. Heterologous 3. In Vivo Evidence of Desensitization. Given the desensitization of GLP-1R occurs at least in part via the clinical interest in the therapeutic benefits of achieving second messenger kinases, cAMP-dependent PKA and sustained chronic elevations of GLP-1R agonists in the PKC (Widmann et al., 1996b; Rajan et al., 2015). Direct plasma through repeated or continual administration, activation of PKC by PMA markedly reduced the ampli- the scope of GLP-1R desensitization has direct thera- tude of GLP-1R–mediated calcium responses, whereas peutic relevance. Up to now, only a few studies have inhibitors of PKC slowed desensitization and increased addressed the relevance of GLP-1R desensitization the duration of calcium transients (Gromada et al., 1996). in vivo. Chronic or intermittent intracerebroventricular Using fusion proteins of wild-type and mutant GLP-1R GLP-1 administration inhibited food intake and re- C-terminal tails, in vitro phosphorylation experiments duced weight gain in rats (Davis et al., 1998). Similarly, revealed that PKC-mediated phosphorylation occurred chronic intermittent administration of GLP-1R agonists predominantly at residues 431/432, and, whereas po- to diabetic rodents is associated with inhibition of food sitions 444/445 and 451/452 could be phosphorylated intake, improvement of glycemia, and reduction in by PKC, they were poor substrates. In these studies, hemoglobin A1c (HbA1c), demonstrating that repeated the serine doublet 441/442 was not a substrate for PKC administration of GLP-1 is not associated with a dimin- (Widmann et al., 1996a). In contrast, studies performed ished therapeutic response in vivo (Szayna et al., 2000; in intact COS-7 cells showed all four of these doublets Rolin et al., 2002; Kim et al., 2003). could be phosphorylated following PKC activation by Considering the prolonged activity and stability of PMA. This could be attributed to different isoforms of GLP-1 mimetics compared with native GLP-1 (Young PKC in intact cells or the possibility that other kinases et al., 1999), it seems likely that islet cells and downstream of PKC activation may be important for extrapancreatic GLP-1R would be exposed for a greater PMA-induced GLP-1R phosphorylation. Phosphoryla- period of time with these ligands than the endogenous tion of at least two of these serine doublets was required peptides. In vitro studies showed that exendin-4 is more to engender PMA-induced heterologous desensitization, potent than GLP-1 in producing GLP-1R desensitiza- with removal of any pair of doublets leading to receptors tion; however, chronic exposure to exendin-4 in normal that were completely resistant to PMA-induced GLP-1R or transgenic mice that express exendin-4 was not desensitization (Widmann et al., 1996a). associated with significant downregulation of GLP- PKA has been implicated as a mediator of desensiti- 1R–dependent responses coupled to glucose homeosta- zation and downregulation of GLP-1R from the cell sis (Baggio et al., 2004). Furthermore, patients treated 990 de Graaf et al. with twice-daily exendin-4 or once-weekly liraglutide Studies performed in CHO and CHL cells recombi- continue to exhibit a decrease in HbA1c and marked nantly expressing GLP-1R revealed internalization via reductions in postprandial glycemic excursion (Buse clathrin-coated pits, although these studies indicated et al., 2004; de Wit et al., 2014). Therefore, although that there might not be complete internalization via in vitro experiments clearly demonstrate that GLP-1R this method (Widmann et al., 1995; Vazquez et al., has the capacity for desensitization, there is little evi- 2005). Studies in CHL fibroblasts demonstrated that dence that it undergoes clinically meaningful desensiti- the same three phosphorylation sites linked to homol- zation in vivo in terms of glucose regulation. ogous desensitization of GLP-1R–mediated cAMP re- There is, however, some limited evidence for de- sponses (441/442, 444/445, and 451/452) are also sensitization occurring in vivo (Nauck et al., 2011). important for internalization, supporting a correlation Administering native GLP-1 continuously into healthy between phosphorylation at these sites and internali- human subjects for 8.5 hours, followed by assessment zation (Widmann et al., 1997). However, the exact of glucoregulatory responses to liquid test meals given contribution of each phosphorylation site to desensiti- 5 hours apart with ongoing continuous GLP-1 infusion, zation of the cAMP response and internalization is demonstrated a reduced ability of GLP-1 to suppress different, indicating that the precise molecular basis gastric emptying and glucagon levels by the second test for control of receptor desensitization and endocytosis meal. In addition, levels of , a is, at least in part, distinct. In addition to phosphoryla- marker of vagal activation, were not as inhibited during tion of these serine doublets, a region of the C-terminal the second test meal compared with the first. However, tail close to the bottom of TMD7 is also of importance in C-peptide and insulin levels were preserved with only a mediating agonist-dependent GLP-1R internalization small reduction in the second meal. These studies reveal (Vazquez et al., 2005). that even short-term continuous GLP-1R stimulation Traditionally for GPCRs, b-arrestin recruitment tar- may be associated with some degree of rapid tachyphy- gets receptors for clathrin-mediated internalization laxis, most evident in effects mediated through the vagus (Ferguson, 2001). However, the literature indicates that nerve and gastric emptying (Nauck et al., 2011). Despite this may not be the case for GLP-1R in some cell back- this evidence for in vivo desensitization, the physiologic grounds, despite the ability of the receptor to interact significance of this is still unclear. with these proteins (Syme et al., 2006). Although there is 4. Receptor Internalization. GLP-1R rapidly inter- little literature on the role that b-arrestin 2 plays in GLP- nalizes as a complex associated with its bound ligand. 1R internalization and desensitization, there is some This has been observed for GLP-1, exendin-4, liraglutide, evidence that these processes can occur independently of and compound 2 (Widmann et al., 1995; Kuna et al., b-arrestin recruitment, in a clathrin-independent mech- 2013;Roedetal.,2014).InBRIN-BD11cells,antibody- anism. Studies performed in HEK293, COS-7, and MIN6 labeled GLP-1R and fluorescently labeled GLP-1 were cells exhibited caveolin-1 regulation of internalization colocalized at the perinuclear space following internali- (Syme et al., 2006; Thompson and Kanamarlapudi, 2015). zation (Kuna et al., 2013). Furthermore, internalization GLP-1R contains a classic caveolin-1–binding motif of fluorescently labeled GLP-1, exendin-4, and liraglu- within the second intracellular loop such that it interacts tide has been observed in both recombinant cell systems and colocalizes with the protein intracellularly. Occur- and primary mouse pancreatic islets (Roed et al., 2014). rence of this interaction was also supported by studies There is also some evidence of ligand-directed bias with using inhibitors of caveolin-1. This mechanism of GLP- GLP-1R internalization, although this has not been 1R internalization has been proposed to occur through directly quantified. The potencies for internalization by activation of Gaq proteins, followed by activation of PKC GLP-1 and exendin-4 were 10-fold higher than liraglu- (Thompson and Kanamarlapudi, 2015). In support of tide, although kinetics of internalization were similar for b-arrestin–independent internalization, it was shown all three ligands (Roed et al., 2014). that knockdown of b-arrestin 1 in INS-1 cells had no Currently, the mechanism for GLP-1R internalization effect on internalization or desensitization of GLP-1R in vivo is unclear, as there is evidence that this mecha- (Sonoda et al., 2008). nism may be cell-type dependent (Widmann et al., 1995; Although internalization can contribute to desensiti- Vazquez et al., 2005; Syme et al., 2006; Thompson and zation of receptors, internalization and desensitization Kanamarlapudi, 2015). As GLP-1R is expressed in multi- are two distinct phenomena. Internalization has the ple tissues throughout the body with distinct physiologic potential to contribute to termination of some GLP-1R– functions depending upon the location of the receptor, this mediated signaling events; however, it does not termi- implies that the mechanism and role of receptor internal- nate all components of this signaling (Kuna et al., 2013; ization and desensitization may be tissue-dependent. In Roed et al., 2015). A recent study revealed that inhibition different cell backgrounds, two distinct mechanisms of of internalization using a dominant-negative form of internalization have been observed, with both mecha- dynamin (K44E) resulted in decreased cAMP forma- nisms dependent on dynamin (Widmann et al., 1995; tion, ERK1/2 phosphorylation, and calcium signaling, Syme et al., 2006; Kuna et al., 2013). suggesting that at least some of the signaling mediated Glucagon-Like Peptide-1 and Its Receptors 991 by these pathways occurs via internalized receptors. In determine the activities of the peptide and its analogs addition, inhibition of GLP-1R internalization also through the biologic actions of the protein. There are attenuates insulin secretion, confirming that internal- traditional bioassays (Table 2) to determine receptor- ization is an integral part of the signaling process for binding affinity and activation of the receptor, and several this receptor (Kuna et al., 2013). Using fluorescently unique methods have been reported for assessing molec- labeled ligands and receptors, studies in recombinant ular recognition and biochemical consequences. In addi- systems displayed colocalization of internalized GLP- tion, molecular imaging of the receptor has demonstrated 1R with adenylate cyclase in (Kuna et al., a potential for noninvasive monitoring of pancreatic b 2013). Taken together, these findings all imply that cells, which may be of great value in the diagnosis and GLP-1R continues to signal following internalization, prognosis of diabetes, tracking the disease progression, and that internalization is not necessarily part of the and evaluating therapeutic interventions, including islet mechanism for desensitization. This level of spatio- regeneration and transplantation. temporal control achieved by the compartmentaliza- tion of signaling through internalization may be vital B. Other Bioassays to specific, fine-tuned responses from GLP-1R activa- 1. Receptor Trafficking. Endocytosis of GPCRs and tion and may have substantial implications both physi- postendocytic trafficking between recycling and endo- ologically and therapeutically for targeting this receptor somal degradation are fundamental mechanisms that in disease management. control the signaling capacity of receptors. Syme et al. 5. Receptor Resensitization. GLP-1R resensitization (2006) studied the trafficking of green fluorescent has not been extensively studied. Following desensiti- protein–fused GLP-1R stably expressed on HEK293 zation, GLP-1R–mediated calcium signaling resensi- cells in response to a GLP-1 agonist. It was found that tizes after removal of extracellular ligand within 1 hour GLP-1R associates with caveolin-1 in the lipid rafts of and does not require de novo protein synthesis (Gromada the cell membrane of HEK293 cells transfected with the et al., 1996). After internalization, GLP-1R colocalizes green fluorescent protein–GLP-1R plasmid and MIN6 with transferrin, a marker for recycling endosomes, cells endogenously expressing the GLP-1 receptor. With suggesting that it is recycling GLP-1R that is returned confocal fluorescence microscopy and immunoprecipi- back to the cell surface after internalization (Roed et al., tation techniques, it was observed that stimulation by 2014). Moreover, there is evidence in HEK293 cells that an agonist induces rapid and extensive internalization GLP-1R recycles back to the cell surface in response to of the receptor in association with caveolin-1, and nearly GLP-1, exendin-4, and liraglutide, even after prolonged all of the receptor was internalized after 15 minutes. agonist treatment. However, recycling was two to three Similarly, Roed et al. (2014) combined microscopy times slower for exendin-4 and liraglutide compared with and a time-resolved fluorescence resonance energy GLP-1 (Roed et al., 2014). In the presence of GLP-1, transfer–based technique for monitoring both receptor receptors were found in transferrin-positive endosomes trafficking and signaling in living cells. The GLP-1 up to 45 minutes post ligand addition, whereas in the receptor rapidly and repeatedly internalizes and recy- presence of exendin-4 and liraglutide colocalization of cles upon ligand activation. GLP-1R recycling was internalized GLP-1R and transferrin was observed for up found to be two to three times slower when induced by to 60 minutes following ligand addition. Resensitization exendin-4 and liraglutide, compared with GLP-1 at is important, as irreversible desensitization would leave equipotent concentrations. Independent of the ligand, a cell unable to respond to external stimuli, and therefore activated receptors demonstrated cycling for a prolonged this mechanism protects against prolonged desensitiza- period of time as well as sustained cAMP signaling. tion (Ferguson, 2001). Nonetheless, to date, the mecha- 2. Immunoassays. Determination of , espe- nism of GLP-1R resensitization remains largely unknown cially endogenous ones, by immunologic assays is consid- and requires further investigation. ered difficult because differential processing of precursor There is also evidence of partial degradation of GLP- molecules gives rise to a number of different peptides that 1R upon prolonged activation with exendin-4, liraglu- may crossreact with antisera raised against GLP-1 tide, and GLP-1, with colocalization observed between (Deacon and Holst, 2009). Subsequent degradation of the receptor and lysosomes (Kuna et al., 2013; Roed the peptides by DPP-4 and NEP 24.11 further compli- et al., 2014). This mechanism leads to downregulation of cates the assay process (Heijboer et al., 2011). receptor numbers. When the full processing pattern of proglucagon was not fully elucidated, the assays were directed to the IV. Pharmacological Tools middle region of GLP-1 and became independent to the processing pattern. This caused peptides with a deletion A. Traditional Bioassays or extension at either the N or C terminus to be detected As GLP-1 exerts its vital physiologic and pharmaco- in addition to GLP-1 itself. Antisera were then made logical functions through interacting with its receptor toward either the C terminus (Hendrick et al., 1993; on the cell membrane, it is important to measure and Holst et al., 1994; Ørskov et al., 1994) or the N terminus 992 de Graaf et al.

(Gutniak et al., 1996). However, it was found that emission-computed tomography (SPECT), and optical C-terminally directed antisera reacted with peptides absorption or fluorescence spectroscopy and imaging, truncated or extended at the N terminus and could not have shown promises in visualizing and quantitating distinguish the full-length GLP-1 from the metabolite molecular targets (Souza et al., 2006). However, assess- resulting from DPP-4 degradation. Similarly, N-terminally ing BCM in vivo has been challenging because the b directed antisera could not differentiate the intact peptide cells that exist in pancreatic islets are small (50–300 mm from truncated or extended peptides at the C terminus. in diameter), scarce (1–2% of pancreatic mass), and Kirk and coworkers developed a sandwich enzyme- scattered throughout the organ (Ueberberg et al., 2009). linked immunosorbent assay for determining exoge- As nonselective imaging modalities like magnetic res- nous GLP-17–36 amide in plasma samples collected from onance imaging and computed tomography have failed to pharmacokinetic studies (Pridal et al., 1995). It is offer a reliable detection method, b cell–specific bio- referred to as active GLP-1 assay. This experiment markers have been investigated and evaluated (Shiue employs an N-terminally directed antibody (polyclonal et al., 2004; Souza et al., 2006; Saudek et al., 2008; rabbit antibody directed to the residues at position 7– Malaisse et al., 2009; Moore, 2009; Ichise and Harris, 14) and a C-terminally directed antibody (monoclonal 2010). Although a number of potential candidates for b mouse antibody directed to the residues at position 26– cell imaging have been reported, such as vesicular mono- 33). When compared with a radioimmunoassay employ- amine transporter, GLP-1R, SUR1, glucose transporter 2, ing a polyclonal rabbit antibody directed to an epitope in glycogen, zinc transporters, fluorodithizone, and mono- the midregion of the peptide, it was found to be superior clonal antibodies, many did not show promising results due to reduced crossreactivity with GLP-1 fragments. It due to either low expression levels or insufficient b cell has a working range from 10 to 500 pmol/L. specificity (Murthy et al., 2008; Schneider, 2008; Moore, 3. Pancreatic b Cell Regeneration. Besides insulin 2009). Vesicular monoamine transporter is expressed at secretion from the pancreatic b cells in a glucose- dopamine nerve ends in the CNS and pancreatic b cells, dependent manner, GLP-1 can induce regeneration of and its ligands, 11C- and 18F-labeled dihydrotetrabena- the cells evidenced by immunohistochemical staining zine derivatives, have demonstrated a potential for b cell studies (Edvell and Lindstrom, 1999; Xu et al., 2006). imaging (Souza et al., 2006; Harris et al., 2008; Kung PDX-1 is essential for pancreogenesis, pancreatic cell et al., 2008). However, controversial findings of nonspe- differentiation, and maturation. Perfetti and coworkers cific binding of [11C]-dihydrotetrabenazine in human demonstrated that continuous infusion of GLP-1 to pancreas raised questions for its clinical application young and old rats upregulates PDX-1 expression in (Goland et al., 2009). islets and total pancreas (Hui and Perfetti, 2002). They Compared with other b cell biomarkers, GLP-1R also found induced pancreatic cell proliferation and b showed promise because of its highly specific expression cell neogenesis (Perfetti et al., 2000). in b cells and strong interaction with its ligands (Tornehave et al., 2008). However, its endogenous ligand, GLP-1, is C. Molecular Imaging difficult to be used as an imaging agent because of its Pancreatic b cells play an important role in glucose extremely short half-life (II. Glucagon-Like Peptide-1) homeostasis, and their destruction is well documented resulting from rapid metabolic degradation. This empha- with diabetes, in particular type 1. Whereas several sizes that b cell imaging via GLP-1R as a biomarker clinical parameters, such as plasma glucose, glycated requires GLP-1 analogs that have high metabolic stabil- HbA1c, insulin, and C-peptide levels, indicate onset and ity as well as strong binding affinity. progress of the disease, the majority of the b cells are Exendin-3 and exendin-4 are found to be resistant to destroyed by the time the symptoms appear (Matveyenko DPP-4 degradation, and these stable GLP-1R agonists and Butler, 2008). Late diagnosis of the disease results have been developed for b cell imaging (Gotthardt et al., from the remaining b cells compensating for the loss of 2006; Wild et al., 2006, 2010; Brom et al., 2010; Reiner insulin production due to cell death, and abnormalities in et al., 2010; Wu et al., 2011; Connolly et al., 2012). Al- blood glucose concentrations are not typically observed ternatively, bicyclic GLP-1 analogs developed by Ahn and until b cell mass (BCM) is diminished by more than 50% coworkers have also demonstrated outstanding results in (Souza et al., 2006). Thus, noninvasive imaging of BCM determining BCM (Ahn et al., 2011; Gao et al., 2011). A would provide accurate and in-time status as well as metal chelator, such as DOTA, NOTA, or DTPA, was useful prediction. b cell imaging has potential in monitor- conjugated to these peptides and subsequently labeled ing the disease progression, evaluating efficacy of thera- with a radiotracer (e.g., 64Cu, 68Ga, 99mTc, and 111In) for peutic interventions in preserving and restoring BCM, PET- and SPECT-imaging experiments. and following up b cell replacement therapies such as islet A SPECT probe was developed based on exendin-4, 40 111 transplantation (Halban, 2004). [Lys (Ahx-DTPA- In)NH2]-exendin-4, to target GLP- Imaging modalities, such as positron emission tomog- 1R for imaging insulinoma in Rip1Tag2 transgenic mice raphy (PET), magnetic resonance imaging, as well as and showed high tumor uptake (Wild et al., 2006; Wicki other nuclear imaging techniques like single-photon et al., 2007). Although imaging agents derived from Glucagon-Like Peptide-1 and Its Receptors 993

TABLE 2 Summary of traditional biologic assays for GLP-1 analogs

Molecular/Cellular Measurement Description/Outcome References Receptor-binding assays: Radioligand competition Determines binding affinity (IC50 or Kd)by Mathi et al., 1997; Tibaduiza et al., 2001; Xiao measuring radioactivity remaining on the et al., 2001 receptor after competitive inhibition of radioligand with a GLP-1 analog. Time-resolved fluorescence resonance Determines binding affinity (IC50) by measuring Maurel et al., 2008; Zwier et al., 2010; Roed energy transfer (FRET) a decrease in FRET signal between Tb-labeled et al., 2014 receptor and fluorescent ligand after competitive inhibition with a GLP-1 analog. Circular dichroism and fluorescence Determines binding affinity by measuring Runge et al., 2007 spectroscopies conformational changes of a receptor protein upon ligand binding. Isothermal titration calorimetry Determines thermodynamic parameters of Wiseman et al., 1989; Bazarsuren et al., 2002; binding, such as the dissociation constant, Donnelly, 2012 enthalpy change, entropy change, and reaction stoichiometry by measuring heat changes during receptor–ligand interaction. Total-internal reflection fluorescence Determines equilibrium constants and Fox et al., 2009; Myers et al., 2012 imaging dissociation rates by monitoring fluorescence- labeled single molecule on lipid bilayer. Surface plasmon resonance Determines dissociation constants by real-time Bazarsuren et al., 2002; Schroder-Tittmann measurement of receptor–ligand interaction. et al., 2010; Locatelli-Hoops et al., 2013 Photoaffinity labeling Identifies residues of peptide and receptor at Chowdhry and Westheimer, 1979; Ji et al., 1997; binding interface that are spatially proximal Vodovozova, 2007; Chen et al., 2009; Miller to each other. et al., 2011 Receptor functional assays: Homogeneous time-resolved fluorescence HTRF technology is an immunoassay based on a Gesellchen et al., 2006; Einhorn and (HTRF) or alpha screen cAMP assays FRET between a Tris-bipyridine europium Krapfenbauer, 2015 cryptate used as a long-lived fluorescent donor and a chemically modified allophycocyanin used as acceptor. Alpha technology is a bead- based proximity assay. Radioimmunoassay Determines receptor-stimulating potency (EC50) Farmer et al., 1975; Wheeler et al., 1995 by quantitative analysis of cAMP production with immobilized anti-cAMP or anti-cGMP antibodies and radiolabeled cAMP/cGMP. FRET-based cAMP assay Determines receptor-stimulating potency (EC50) Holz, 2004; Nikolaev et al., 2004; Landa et al., by measuring a decreased FRET signal 2005; Harbeck et al., 2006; Sloop et al., 2010 between fluorescent proteins (CFP and YFP) and Epac protein. Luciferase reporter assay Determines receptor-stimulating potency (EC50) Grynkiewicz et al., 1985; Cullinan et al., 1994; by measuring luminescence that is increased Bode et al., 1999; Miranda et al., 2008; Murage by transcription of transfected luciferase et al., 2008; Smale, 2010 reporter plasmid linked to cAMP response element. Intracellular calcium ion Determines receptor activation by measuring Grynkiewicz et al., 1985; Cullinan et al., 1994; intracellular Ca2+ level with calcium-sensitive Bode et al., 1999 dye, Fura-2. Determination of incretin effects: Glucose tolerance test (GTT; oral GTT, Determines insulinotropic action of GLP-1 Kreymann et al., 1987; Toft-Nielson et al., 1996 OGTT; intraperitoneal GTT, IPGTT; analogs by measuring glucose level after their intravenous GTT, IVGTT) administration. Insulin secretion Determines potency of GLP-1 analogs by Albano et al., 1972; Andersen et al., 1993; Goke measuring insulin secretagogue action. et al., 1993b; Toft-Nielson et al., 1996; Kjems et al., 2003; Peyot et al., 2009 these stable GLP-1R agonists showed a high renal visualize autologous islets that have been transplanted uptake, the potential of GLP-1R in visualizing b cells into human muscle, proving a clinical potential of human has been demonstrated by PET and SPECT imaging of b cell imaging via GLP-1R (Pattou et al., 2010). insulinoma with radiolabeled exendin-3 and exendin-4 In addition to GLP-1R agonists described above, (Gotthardt et al., 2006; Brom et al., 2010; Wild et al., antagonists, such as exendin9–39, were also developed 2010). As an excellent PET tracer, 64Cu was used to label as b cell imaging agents (Mukai et al., 2009). Radiola- exendin-4, and the resulting 64Cu-labeled exendin-4 beled at residues with 125I-Bolton-Hunter re- 64 40 analog ( Cu-DO3A-VERSUS-Cys -exendin-4) demon- agent, exendin9–39 was examined for receptor specificity strated a feasibility for in vivo imaging of intraportally in vitro and in vivo. Accumulation of radioactive signals transplanted islets in mice by showing high and spe- in b cells was observed, although the resolution of the cific uptake in INS-1 tumors despite substantial renal imaging technique was low. uptake (Wu et al., 2011). Remarkably, [Lys40(Ahx- Ahn and colleagues have developed PET-imaging 111 DTPA- In)NH2]-exendin-4 was successfully used to agents based on the GLP-1 sequence to quantitate BCM 994 de Graaf et al.

(Ahn et al., 2011; Gao et al., 2011). As the endogenous displayed reduced levels of fasting glucose and HbA1c, as GLP-1 suffers from rapid metabolic degradation, they well as improved insulin sensitivity and b cell function. introduced two lactam bridges to GLP-1, one in the The proof-of-concept for the use of GLP-1 in diabetes dem- N-terminal region and the other near the C terminus. onstrated in this study was clear, yet major challenges These two strategically placed lactam bridges were found existed that involved acceptable modes of administration to protect the cyclic peptides from proteolytic cleavages by and the need to improve duration of hormonal action. NEP 24.11 (Murage et al., 2010). In addition, Ala at There are now a wide variety of different peptidic position 8 was replaced with D-Ala to prevent the DPP-4 GLP-1R agonists with variable durations of action and degradation. These modifications not only enhanced different advantages and disadvantages for clinical use. metabolic stability of the GLP-1 analog, but also increased The first GLP-1R agonists to enter clinical use were pep- binding affinity to the receptor. Dynamic PET scans over tide analogs and derivatives, as exemplified by exendin-4 60 minutes showed a high pancreatic uptake of a bicy- (Eng et al., 2014), liraglutide, , and clic GLP-1 analog, [D-Ala8]-c[Glu18,Lys22]-c[Glu30,Lys34]- (Table 3). Unfortunately, these peptides require paren- 64 68 GLP-17–36, labeled with Cu or Ga, which disappeared teral administration, due to instability in the proteolytic in streptozotocin-induced type 1 diabetic mice or was com- milieu of the digestive tract, making it impossible to petitively displaced by coinjection of unlabeled exendin-4 deliver adequate drug reproducibly via oral route. Tech- (Manandhar et al., 2013). Ex vivo scans and histology nology has advanced so much, however, that easy-to-use were also carried out to further confirm the PET-imaging pens for s.c. administration has become quite common. findings (unpublished data). is a potent peptide that was recently tested with s.c. administration in a phase 2 clinical study of 411 patients (Nauck at al., 2016), showing improvement V. Pharmaceutical Development in HbA1c levels by 1.7%. This peptide is now being and Therapeutics studied for oral dosing. This field has progressed so As noted above, GLP-1 is a gastrointestinal peptide quickly that there are now even devices that allow hormone that is secreted from L cells scattered in the longer-term activity being developed. Intarcia is an distal small intestinal mucosa. It is released physiolog- implantable device that releases exenatide over 6– ically when ingested nutrients reach that level of the 12 months. This has been studied in two phase 3 trials bowel, typically reflecting a meal large enough to benefit (Mullard, 2014). from the incretin effect of this hormone. Among its other We have finally entered an era in which small- physiologic effects is the slowing of gastric emptying, molecule nonpeptidic GLP-1R agonists are now available again reflecting the benefit of titration of the rate of (Fig. 7), although it still poses a tremendous challenge to delivery of nutrients to the absorptive surface so as not to make them adequately potent and bioavailable (Yang overwhelm the absorptive capacity of the intestine. This et al., 2015a). Most of these are thought to act within the peptide has a very short half-life in the circulation, with helical bundle of GLP-1R. Crystallization of two mem- , particularly by DPP-4, and renal clearance bers of the class B GPCR subfamily in 2013 (Hollenstein having prominent roles. This endoprotease cleaves the et al., 2013; Siu et al., 2013) provided a clue to why de- amino-terminal dipeptide from endogenous GLP-1 pep- velopment of nonpeptidic modulators has been so diffi- tides; GLP-17–36 amide or GLP-17–37 is cleaved to yield cult. Unlike the class A GPCRs that have tight helical GLP-19–36 amide and GLP-19–37, respectively, which are bundles with a well-defined pocket for small-molecule four orders of magnitude less active than the natural ligands, the class B helical bundles appear to be much hormone (Montrose-Rafizadeh et al., 1997). Recognition more open without defined small-molecule docking sites. of this led to the earliest strategy to increase endogenous Recently, one of the small-molecule agonists (BETP) had GLP-1 levels using DPP-4 inhibitors, rather than acting its site of action determined as covalently binding to a at GLP-1R. This was ultimately followed by the devel- cysteine residue in intracellular loop 3 on the cytosolic opment of molecules having intrinsic GLP-1R agonist side of the plasma membrane (Nolte et al., 2014), a mo- activity. As described below, most of these are peptides lecular mechanism that was a major surprise, without and their derivatives, but small-molecule agonists have precedent in other members of this subfamily. A mole- also surfaced. All of them have potential importance for cule from TransTech Pharma, TTP054, is presently diabetes, obesity, and associated metabolic and cardio- undergoing phase 2 clinical trials (Mullard, 2014). vascular complications. Other candidate applications are also discussed. A. Peptidic Analogs The earliest recognition that GLP-1 exhibited effects 1. GLP-1 Analogs. Several GLP-1 analogs have suc- useful in the management of diabetes came from rodent cessfully reached the market and become an important studies (Gutniak et al., 1992; Nauck et al., 1993c). This treatment of obesity and T2DM today (Table 3). Several was followed by a seminal study in which GLP-1 itself reviews have been published during the past that de- was administered s.c. over a 6-week period in patients scribe the approved and emerging GLP-1R agonists and with T2DM (Zander et al., 2002). These patients also summarize the most important clinical trials as well Glucagon-Like Peptide-1 and Its Receptors 995 as toxicological considerations (Lund et al., 2011; Madsbad approved by the FDA in 2014 for once-weekly dosing of et al., 2011; Garber, 2012; Meier, 2012; Montanya, 2012; 0.75 mg s.c. (Glaesner et al., 2010). Lorenz et al., 2013; Trujillo and Nuffer, 2014; Trujillo There is still a need for optimization of once-weekly et al., 2015; Madsbad, 2016; Zaccardi et al., 2016). GLP-1 analogs because both albiglutide and dulaglutide (BIM-51077; Roche, Basel, Switzerland) are less efficacious than liraglutide with respect to weight is a potent GLP-1 analog with 93% homology to human loss (Dungan et al., 2014; Pratley et al., 2014). GLP-17–36 amide as it has two amino acid substitutions in The next generation of GLP-1 analogs using the revers- positions 8 and 35 with aminoisobutyric acid (Aib). ible albumin affinity is represented by semaglutide (Novo Taspoglutide is protected against DPP-4 degradation by Nordisk, Bagsværd, Denmark) currently in phase 3 clini- 8 35 8 26 34 the Aib /Aib substitution but is still cleared rapidly cal trials. This analog Aib Lys Arg -GLP-17–37 has a by the kidney. A sustained-release formulation that more complex side chain composed of hexadecandinoyl contains zinc-chloride and precipitates as a sub cutis attached to Lys26 via an L-g-glutamicacidlinkeranda deposition was developed. This once-weekly dosing for- small hydrophilic spacer. The Aib8 was introduced to mulation of taspoglutide has shown promising antidia- improve the DPP-4 stability beyond that of liraglutide, betic efficacy in clinical trials. Unfortunately, the analog and the new side chain increased the albumin affinity, was discontinued in phase 3 clinical trials due to severe resulting in a half-life in humans long enough for gastrointestinal side effects (Retterstol, 2009; Dong et al., once-weekly dosing. In a phase 2 study over 12 weeks, 2011; Garber, 2012). semaglutide dose-dependently reduced HbA1c and The GLP-1 human serum albumin fusion protein, body weight, with higher doses being more effective albiglutide, also known as albugon, discovered by than liraglutide (Nauck and Sesti, 2012; Trujillo and Genentech (South San Francisco, CA) and developed Nuffer, 2014; Lau et al., 2015). by GlaxoSmithKline (Brentford, UK), was approved by 2. Exenatide Experience. In 1993, exendin-4 was iso- the Food and Drug Administration (FDA) in 2014. To lated from the salivary glands of the have a free N-terminal, which is important for GLP-1 (Heloderma suspectum) and found to be highly active activity, the peptide was fused via the C terminus to on GLP-1R (Goke et al., 1993a). Exendin-4 is a 39– albumin. The construct is composed of a tandem repeat amino-acid peptide that shares approximately 50% 8 8 of Ala to Gly GLP-17–36 that is fused to the N terminus amino acid sequence identity with human GLP-1. of human albumin. Gly8 GLP-1 was used to protect for The discovery of the antidiabetic effects of exendin-4 DPP-4 degradation of the N terminus. The plasma half- increased the interest in this peptide, and several life was extended to about 6–8 days, which made this pharmaceutical companies thus focused on its clinical fusion construct applicable for once-weekly dosing. One development as well as on discovery efforts in exendin- drawback relates to a significantly reduced potency that based GLP-1 analogs. was probably due to a combination of the Gly8 modifi- In 2005, the first GLP-1R agonist exenatide (AstraZeneca, cation and the covalent attachment to the larger protein Cambridge, UK) was approved by the FDA for treat- human serum albumin. The tandem repeat of GLP-1 ment of T2DM. Exenatide, as well as other GLP-1R was used to obtain longer distance between albumin agonists, reduces blood glucose through multiple mech- and the distal GLP-1 peptide, but the affinity for GLP- anisms, including enhancement of glucose-dependent 1R is 0.61 nM compared with 0.02 nM of the native insulin secretion, suppression of excess glucagon secre- peptide. The approved initial dose to treat T2DM is tion, reduction of food intake, and slowing of gastric 30 mg given s.c. once per week, which may be increased emptying. It is administered s.c. twice daily with doses to 50 mg on the same day of each week if the glycemic between 5 and 10 mg (Cvetkovic and Plosker, 2007). response is inadequate (Kim et al., 2003; Tomkin, 2009; The very low therapeutic dose of exenatide made it an St Onge and Miller, 2010). excellent candidate for an extended release formula- Dulaglutide (LY2189265; Eli Lilly, Indianapolis, IN) is tion. Pharmaceuticals (San Diego, CA), Eli Lilly, a GLP-1R agonist fused to a modified Fc fragment of and Alkermes (Dublin, Ireland) developed a formulation immunoglobulin that acts as a carrier to secure long to prolong exenatide release, using biodegradable poly- residence time by reduced in vivo clearance. The se- meric microspheres that entrap exenatide. The micro- 8 22 36 quence of the fusion protein Gly Glu Gly -GLP-17–37- spheres consist of exenatide incorporated into a matrix of 234,235 228 (Gly4Ser)3Ala-Ala Pro -IgG4-Fc is modified in the poly (D,L-lactide-co-glycolide), which is a common biode- N terminus of GLP-1 to protect against DPP-4 cleavage gradable medical polymer with a history of safe use in 8 (Gly ), and the IgG4 Fc is modified to reduce interaction humans. The final formulation was approved in 2011 as with high-affinity Fc receptors and avoid antibody- Bydureon for once-weekly dosing (Kim et al., 2007; dependent cell-mediated cytotoxicity. The linker (Gly4- Malone et al., 2009). Ser)3Ala was carefully selected to retain high GLP-1R (Sanofi, Paris, France) is a once-daily affinity as direct fusion dramatically reduced in vitro short-acting GLP-1R agonist used in the treatment of activity by ;95%. The half-life is above 1.5 days in rats T2DM. The discovery of lixisenatide was inspired by the and more than 2 days in monkeys. Dulaglutide was development of structure-inducing probe technology, 996 de Graaf et al.

TABLE 3 Peptidic GLP-1R agonists that have been launched or are in late-stage clinical trials

Analog Description Company Dosing Regimen Status Exenatide Exendin-4 Amylin/Eli Lilly; AstraZeneca Twice daily Launched Exenatide once weekly Exendin-4 Amylin/Alkermes/Eli Lilly; Once weekly Launched AstraZeneca Lixisenatide Exendin-4 analog Sanofi-Aventis Once daily Launched Liraglutide GLP-1 analog linked with a fatty acid Novo Nordisk Once daily Launched Albiglutide GLP-1 analog fused to albumin GlaxoSmithKline Once weekly Launched Dulaglutide GLP-1 analog fused to Fc Eli Lilly Once weekly Launched Semaglutide GLP-1 analog linked with a fatty acid Novo Nordisk Once weekly Phase 3 Semaglutide (NN9924) GLP-1 analog linked with a fatty di-acid Novo Nordisk Oral Phase 2 Taspoglutide GLP-1 analog formulated for sustained release Roche Once weekly Suspended which improves the half-life of structure-inducing a minimal initial burst and constant release are appli- probe conjugates compared with the parent peptide by cable to once-daily dosing (Qian et al., 2009). enhancing stability and, as a consequence, creating resistance to proteolytic degradation. Lixisenatide, des- B. Nonpeptidic Modulators 36 6 Pro -exendin-41-39-Lys amide, comprises 44 amino Even though more than two decades have passed acids and is based on the exendin-4 sequence with a since the cloning of the GLP-1R (Thorens, 1992), very proline deletion in position 36 and C-terminal exten- few nonpeptidyl-based GLP-1R agonists have been pub- sion with six . Lixisenatide has an extended lished. One might speculate on the reason: the ligand– in vivo half-life of 91 minutes after i.v. administration receptor interaction leading to activation requires (in rabbits) compared with 43 minutes for exendin-4. multiple interfaces that are difficult to mimic with a Lixisenatide was approved in 2013 and has a substan- small molecule. The majority of the nonpeptidyl-based tial sustained effect on gastric emptying and post- GLP-1R agonists described in the literature are re- prandial glucose excursions that may be related to the ceptor modulators that bind to allosteric sites and do relatively shorter half-life. The combination with basal not compete with GLP-1 in the orthostatic site. Many insulin to lower fasting plasma glucose is clinically such GLP-1R agonists were only mentioned in patents valuable and could differentiate lixisenatide from and are not included in this section, as their experi- other GLP-1R agonists, especially from those long- mental data have not been peer-reviewed. There are acting ones with little effect on gastric emptying and also small numbers of nonpeptidic GLP-1R antago- postprandial glucose (Werner et al., 2010; Christensen nists, and these will be discussed in the last part of this et al., 2014; Rosenstock et al., 2014). section. One review article is also referred concerning 3. GLP-1 Mimetics. A novel class of 11–amino-acid nonpeptidic GLP-1R modulators (Willard et al., 2012a). GLP-1R agonists was discovered that consist of an To date, only one nonpeptidic GLP-1R agonist has analog of only the first 9 amino acids of GLP-1, in which entered into clinical trials. The compound has not been the remaining 21 amino acids have been truncated and specifically disclosed, but a publication from the com- attached to an unnatural biphenyl diamino acid at the pany described that it stimulated glucose-dependent C terminus. The optimization of the sequence with insulin release in isolated islets from rodents and several unnatural amino acids provided 11-mer GLP-1 improved glycemic control in an oral glucose tolerance mimetics with similar in vitro potency as the natural test (Gustavson et al., 2014). 30-mer peptide. The optimized 11-mers (e.g., BMS- Boc5, a substituted cyclobutane with four chiral 686117) were reported to reduce plasma glucose and centers, is among the few nonpeptidic GLP-1R agonists increase plasma insulin concentrations in diabetic ob/ that have been comprehensively studied. It was discov- ob mice after a single relative high s.c. dose of the ered during a high-throughput screening against 48,160 peptide (Mapelli et al., 2009; Haque et al., 2010). Based small molecules using a luciferase reporter assay in on NMR studies, the structures of these 11-mer peptides HEK293 cells expressing the rat GLP-1R (Chen et al., were determined and used to design lactam- and disulfide- 2007). Boc5 is the only nonpeptidic GLP-1R agonist based cyclic constrained peptides that retained high GLP- that has been shown to compete for 125I-GLP-1 in 1R potency (Hoang et al., 2015). binding assays and to be functionally antagonized by BMS-686117 has a relatively shorter half-life of about exendin9–39, thus acting like the natural peptide ligand. 2 hours in dogs, and, accordingly, a sustained release The antidiabetic effect of Boc5 was demonstrated in formulation is required for clinical use. Formulation various animal models. A 4-week dose-response study with Zn (II) forms a poorly water-soluble adduct such was performed in db/db mice with daily i.p. dosing of that the BMS-686117 content and reversible release of Boc5 at 0.1–3 mg. There was a dose-dependent decline which can be tailored by varying the Zn:peptide ratio. in HbA1c during the treatment that continued to An in vivo study in dogs of an decrease until 2–3 weeks after the last dose. In contrast, optimized sustained release formulation concluded that Boc5 had no effect on HbA1c in nondiabetic C57BL/6J Glucagon-Like Peptide-1 and Its Receptors 997

Fig. 7. Nonpeptidic GLP-1R modulators and peptide mimetics. Liraglutide (Novo Nordisk) was the first approved human GLP-1 analog to teat diabetes (European Union, 2009; United States, 2010) and obesity (United States, 2014; European Union, 2015). Liraglutide has 97% sequence 998 de Graaf et al.

(wild-type) mice. Boc5 also inhibited food intake and increased plasma insulin levels in a glucose tolerance decreased body weight of db/db mice by up to 16% study using Sprague Dawley rats as well as hypergly- during the study with reduced fat mass. In addition, it cemic clamped Spraqgue Dawley rats. One drawback for was demonstrated that Boc5 decreased the elevated further development of both quinoxaline- and pyrimidine- levels of fasting insulin in db/db mice, and the insulin based leads is the chemical instability in the presence of sensitivity was improved after 4 weeks of treatment. nucleophiles (Sloop et al., 2010). Thus, the effects of Boc5 seen both in vitro and in vivo Recently, a tricyclic pyridoindole-based series of suggest that this compound has similar pharmacologi- GLP-1R allosteric modulators was identified by high- cal properties as peptidic GLP-1R agonists. Medicinal throughput screening measuring intracellular calcium chemistry efforts were made to understand the SAR, mobilization in the presence of low levels of GLP-1 or and one analog, WB4-24, was found to be more potent glucagon. These compounds were shown to potentiate in vitro compared with Boc5. WB4-24 was shown to glucose-dependent insulin release in primary mouse islets, have beneficial effects on glucose homeostasis and body but no in vivo studies were described (Morris et al., 2014). weight in diet-induced obese mice (Su et al., 2008; He The potential use of GLP-1R allosteric modulators to et al., 2010, 2012; Liu et al., 2012). enhance the effect of endogenous GLP-1 was studied After an unsuccessful screening of 500,000 discrete in vivo. An i.v. glucose tolerance study was carried out in small molecules with a GLP-1 competitive binding assay, which coadministration of BETP to rats dosed with a substituted quinoxaline was identified in subsequently subsaturating concentrations of oxyntomodulin mark- performed functional screening of 250,000 compounds. edly increased the insulinotropic effect of oxyntomodu- This hit as well as some closely related analogs activated lin (Willard et al., 2012b). the formation of cAMP in BHK cells expressing the GLP-1R (Fig. 7, compounds 1 and 2). However, it did C. Currently Approved Clinical Applications not compete with 125I-GLP-1 in a binding assay, but A number of GLP-1R agonists have been approved for augmented the binding of the tracer. Furthermore, its clinical use to improve glycemic control in adults with functional effect could not be antagonized by exendin9–39. T2DM. Diet and exercise are the expected initial approach Functionality counter-screening analysis showed that to treat these patients, with GLP-1R agonists prescribed the quinoxalines were inactive in BHK cells expressing as second-line drugs for patients who are refractory to the GIPR, GLP-2 receptor, and GCGR. They were capable of current standard of care. GLP-1R agonists have no cur- releasing insulin from wild-type mouse islets, but not rent role in the management of patients with type 1 from islets isolated from GLP-1R knockout mice. These diabetes, and should be avoided in those patients having a quinoxalines were thus concluded to be selective GLP-1R history of or medullary thyroid . The ago-allosteric modulators. Further SAR studies around use of this category of drugs in T2DM reflects their direct these initial leads as well as some of the later identified effects on the b cells within the pancreatic islets to nonpeptidic modulators have all been published (Knudsen increase insulin biosynthesis and secretion, to stimu- et al., 2007; Teng et al., 2007; Coopman et al., 2010; Irwin late cell proliferation, and to reduce apoptosis. A key et al., 2010; Koole et al., 2010; Cheong et al., 2012; Li et al., benefit of GLP-1R agonists is the glucose-dependent 2012a). effects on the b cell, with reduction in blood glucose Pyrimidine-based ago-allosteric modulators (Fig. 7, only in patients exhibiting hyperglycemia. This elim- compounds A and B) of GLP-1R were found by screening inates the hypoglycemia risk of many other antidia- of a small library enriched with relevant pharmaco- betic drugs. In fact, GLP-1R agonists have now largely phores for GPCRs. The screening was performed in replaced the use of (e.g., rosiglita- HEK293 cells coexpressing hGLP-1R and a cAMP re- zone). Adverse responses such as peripheral edema, sponse element luciferase reporter. Like the quinoxaline- weight gain, congestive , and osteoporosis based modulators, the pyrimidines did not compete with associated with the latter are not observed in patients 125I-GLP-1 in a binding assay and the functional effect treated with GLP-1R agonists (Drucker et al., 2010). was not antagonized with exendin9–39. They were shown GLP-1R agonists also exhibited cardiovascular benefits, to enhance GLP-1–induced cAMP signaling and insulin such as lowering blood pressure, improving lipid profiles, release in isolated rat islets. Compound B (BETP) and possibly even enhancing cardiac contractility and

homology to human GLP-1 and was designed to reversibly bind to albumin by attachment of palmatic acid via a L-g-glutamic linker to lys26 in Arg34 34 34 34 26 GLP-17–37. The modification of Lys to Arg made it possible to produce Arg GLP-17–37 in yeast, followed by acylation of Lys . The native GLP-1 peptide has a half-life of approximately 2 minutes due to rapid cleavage of GLP-17–37 to GLP-19–37 by DPP-4 (Deacon et al., 1995a,b; Vilsboll et al., 2003). Liraglutide comprises the natural GLP-1 N terminus, but has a half-life of about 11 hours after s.c. dosing to humans combined with a delayed absorption from sub cutis that gives a pharmacokinetic profile applicable for once-daily administration. The reason for extended circulation is dueto reversible albumin binding that protects from DPP-4 degradation and glomerular filtration, whereas the delayed absorption is explained by the ability of liraglutide to form heptamers by self-assemble controlled by the fatty acid side-chain at position 26. Liraglutide is well tolerated and capable of substantially improving glycemic control with low risk of hypoglycemia and weight loss benefit (Knudsen et al., 2000; Knudsen, 2004; Madsen et al., 2007; Steensgaard et al., 2008; Dharmalingam et al., 2011; Wang et al., 2015). Glucagon-Like Peptide-1 and Its Receptors 999 endothelial function. In addition to its action on islet b It is curious whether the induction of , which is cells, this hormone exerts effects on the following: 1) believed to be a result of slowed gastric emptying, is pancreatic a cellstoreduceglucagonsecretionandhepatic similar in the short-acting and longer-acting GLP-1R glucose output; 2) the cardiovascular system to elevate agonists, yet this seems to be attenuated quickly in the glucose utilization and protect the heart and vasculature longer duration agonists and may take weeks to months against inflammation; 3) the brain to decrease food intake; to improve for the shorter duration agonists. Such a and 4) the gut indirectly to slow gastric emptying. phenomenon most likely reflects differences in desensi- 1. Selection. As the number of GLP-1R agonists in tization of GLP-1R, but the underlying mechanism has clinical use and trials expands, it will become clearer not been carefully studied. Although the weight loss effect whether there are advantages of one drug over another seen with some of these GLP-1R agonists was thought in a specific clinical setting. We have already observed to be related to slowed gastric emptying, the long-acting differences in the frequency of nausea, presumably agents seem to result in greater weight reduction than reflecting the reduced gastric emptying, among these the short-acting ones, suggesting certain other mecha- drugs. There is also variation in the pathway of inacti- nism is dominant. This might relate to reduced appetite, vation and excretion of these drugs, making the selec- potentially following GLP-1 action in the hypothalamus tion of agent in patients with renal insufficiency or other CNS regions. important. Although biased agonism is now recognized 2. Efficacy. The first phase 2 trial to study a GLP-1R for some of these agonists, there is still insufficient agonist in T2DM was started in 1999, comparing twice- information on the broad pharmacodynamics properties daily administration of exenatide to saline (Kolterman of these drugs to differentiate the contribution of biased et al., 2003). Not only was the exenatide effective in agonism from pharmacokinetic behavior to differential reducing glucose levels, but it did not induce hypogly- clinical responses, side effects, or toxicities, although cemia in patients who were on insulin therapy. Al- this is likely to be an important consideration in future though exenatide was approved by the FDA for use in therapeutic development. diabetes in 2005, concerns about hypoglycemia in insulin- A very practical and useful categorization of GLP-1R requiring patients persisted, and this was finally studied agonists is based on their duration of action. The in 2008 (Buse et al., 2011). The dramatic observations shortest-acting agents, like exenatide and lixisenatide of enhanced reduction in HbA1c and no increase in with half-lives of 2–5 hours, seem to exert their hypoglycemic episodes, as well as weight loss, formally beneficial effects predominantly by slowing gastric launched a new series of clinical studies to follow. A emptying and thereby reducing the rate of carbohydrate meta review published in Lancet in 2014 (Eng et al., delivery, digestion, and absorption, leading to normal 2014) chose 15 of 2905 studies that represented 4348 postprandial glucose levels. Of course, other nutrients subjects to evaluate. HbA1c levels were decreased by an also empty more slowly, and this has resulted in lower average of 0.44%, and weight was reduced by an average postprandial levels of free fatty acids and triglycerides, of 3.22 kg, without any increase in hypoglycemic epi- which has some metabolic benefits. For instance, de- sodes. Of note, the levels of HbA1c were not different creased levels of chylomicrons are desired in patients using GLP-1R agonist with boluses of insulin, but the with diabetes or metabolic syndrome. As the duration of frequency of hypoglycemic episodes was significantly GLP-1R agonist action is extended with peptides like reduced with the former. albiglutide, dulaglutide, long-acting release exenatide, Short-acting GLP-1R agonists known to better con- or liraglutide (half-lives of 12 hours to several days), the trol postprandial glucose levels have not been directly impact on blood glucose levels is enhanced as insulino- compared with that of long duration administered with tropic action and activity to reduce glucagon become basal insulin. Each has theoretical advantages and more prominent than the effect on gastric emptying. perhaps should be chosen based on the characteristics Such prolonged activities have also been associated of the patient. Because long-acting agents can be given with increased side effects, such as diarrhea, tachycar- with short-acting insulin at meal time now, another dia, immune responses, and injection site reactions. Of important option exists, but needs to be evaluated. interest, nausea and vomiting appear to have greater The DURATION-3 trial (Diamant et al., 2014) com- incidence with the shorter-acting peptides. The longer- pared long-acting release exenatide administered once acting agents have typically provided more consistent per week to (a long-acting, man-made effects on GLP-1R activation and blood glucose levels. version of human insulin) in 456 patients with T2DM Because the insulinotropic action of GLP-1 is depen- who had not achieved satisfactory glucose control with dent on the presence of hyperglycemia, long duration maximal tolerated doses of oral agents. Exenatide was activity does not increase the risk of hypoglycemia. able to better manage the blood glucose, with HbA1c Therefore, long-acting release formulations have ob- levels being reduced by 1.01%, whereas glargine plus vious advantages if the goal is to lower early morning oral agents only reduced this by 0.81%. Again, the most glucose levels, because they retain efficacy throughout notable observation was a much lower incidence of the night. hypoglycemia in patients taking the GLP-1R agonist. 1000 de Graaf et al.

The AWARD-4 study did use dulaglutide injected being dose- and duration-dependent. Another retrospec- only once per week and rapid-acting insulin with each tive cohort study also showed a reduction in heart failure meal to demonstrate improved levels of HbA1c than in patients with diabetes (Velez et al., 2015). More than were observed in basal and bolus insulin administration 19,000 adult patients with diabetes were studied and (Jendle et al., 2014). Fixed-dose combinations of GLP- matched with twice as many controls, and 1,426 patients 1R agonist and basal insulin have substantial appeal for with diabetes used GLP-1R agonists. This was also convenience, although, at the current time, expense is a encouraging, but only points toward the need for pro- major concern. The DUAL-1 and DUAL-2 studies exhibited spective studies. a 1.9% reduction in HbA1c in patients who followed a There is increasing excitement about the potential titration algorithm with a pen delivery device (Buse et al., role for fixed-dose combinations of a long-acting insulin 2011; Gough et al., 2014). preparation and a GLP-1R agonist as an adjunct to diet 3. Benefits. In addition to its action on blood glucose, and exercise to improve glycemic control in adults with mediated largely by impact on the pancreatic islet b and T2DM. Two such preparations have recently been rec- a cells, GLP-1R agonists have been shown to display ommended for FDA approval by advisory committees, favorable cardiovascular effects. This includes improve- based on portfolios of clinical trials, including double- ments in serum lipids, myocardial contractility, and blinded designs. These include IDegLira from Novo endothelial function (Mudaliar and Henry, 2010). The Nordisk, combining Tresiba () and effects on lipids are likely to reflect slowed gastric Victoza (liraglutide) into a once-daily injectable prod- emptying and reduced delivery for absorption, as well as uct (http://www.fda.gov/downloads/advisorycommittees/ impact on hepatic and fat cell metabolism. Presumably, committeesmeetingmaterials/drugs/endocrinologicand- the direct cardiovascular benefit was achieved via abun- metabolicdrugsadvisorycommittee/ucm502074.pdf), and dant levels of GLP-1R expression in cardiomyocytes and iGlarLixi from Sanofi, combining Lantus (insulin glar- blood vessels (Nolte et al., 2014). Experiments in rodents gine) and Lyxumia (lixisenatide) into a similar prod- showed that GLP-1R agonists can reduce myocardial uct (http://www.fda.gov/downloads/advisorycommittees/ infarct size and improve left ventricular function, in- committeesmeetingmaterials/drugs/endocrinologicand- cluding better wall motility and ejection fractions metabolicdrugsadvisorycommittee/ucm502559.pdf). Both (Nikolaidis et al., 2004b; Noyan-Ashraf et al., 2009). of the component drugs in IDegLira and the insulin There also exists encouraging evidence in rodents of glargine in iGlarLixi are already FDA approved, whereas the neuroprotective role of GLP-1 for AD, PD, and even lixisenatide is currently undergoing independent regu- stroke (Harkavyi et al., 2008; McClean et al., 2011; latory review. The efficacy of both combination products Teramoto et al., 2011), although this has not been was superior to that of the components alone, leading to followedupbyclinicalstudies. the successful outcomes. The safety of them appears to be A recent meta-analysis explored the impact of GLP- consistent with that of the individual components, and 1R agonists on lipids in clinical trials (Sun et al., 2015). there are indications that some side effects like nausea Thirty-five trials with 13 treatments lasting a minimum and weight gain may actually be less in the combination of 8 weeks were included, and GLP-1R agonists were preparations than for some individual components. It associated with modest improvements in total choles- seems likely that at least one of these products or some- terol, low-density lipoprotein cholesterol, and triglycer- thing alike will achieve a prominent role in therapy soon. ide levels, whereas there was no change in high-density lipoprotein cholesterol. It is not yet clear whether this D. Potential Applications for Other Diseases translates into an improvement in cardiac and vascular 1. Neurologic Disorders. The expression of GLP-1R events. It follows that a latest retrospective review of in multiple sites of the nervous system and the in- macrovascular outcomes in patients with T2DM who volvement of GLP-1 in satiety and food intake regula- had been treated with insulin alone or with GLP-1R tion are well-established (II. Glucagon-Like Peptide-1). agonists (Paul et al., 2015) suggested that heart failure, Because this incretin was also shown to improve learning myocardial infarction, and stroke were less common in in rats (Oka et al., 2000) and exert neurotrophic or patients who were taking the drug. Although such neuroprotective effects when given intracerebroventric- epidemiologic studies need to be interpreted carefully, ularly, its potential therapeutic value for AD, PD, and this is inspiring and clearly worthy of prospective eval- has been investigated (During et al., 2003; McClean uation. A nested case-control study was also recently et al., 2011; Gong et al., 2014b). performed (Gejl et al., 2015). T2DM patients who de- AD is characterized by progressive cognitive decline veloped one or more of these cardiovascular problems with a defined neuropathology of Ab production and tau were compared with control patients with diabetes who hyperphosphorylation (Claeysen et al., 2012). T2DM did not develop such cardiovascular events. More than has been identified as a risk factor for AD, and an 10,000 patients were included, and 1,947 had one or epidemiologic study indicated that 65% of T2DM pa- more of these events. Liraglutide was associated with tients showed evidence of featured AD plaques in their significant risk reduction, with the degree of reduction autopsied brains (Bassil et al., 2014), indicating that Glucagon-Like Peptide-1 and Its Receptors 1001 insulin desensitization in the periphery may be a factor such a protective action was observed in rodent models in initiating or accelerating the development of neuro- of PD: GLP-1 and its mimetics were effective to protect degenerative processes. It has led to the notion that tyrosine hydroxylase–positive dopaminergic neurons drugs developed for the treatment of T2DM may be and to preserve dopamine levels in 1-methyl-4-phenyl- beneficial in modifying the pathophysiology of AD. In 1,2,3,6-tetrahydropyridine–treated animals. Treatment preclinical models of AD, GLP-1 decreases Ab toxicity with a GLP-1R agonist resulted in improved motor in vitro (During et al., 2003; Perry et al., 2003). A function, as demonstrated by rotarod and pole tests follow-up study demonstrated that GLP-1 and exendin- (Kim et al., 2009; Li et al., 2009). Recently, a single-blind 4 could protect cultured rat hippocampal neurons of exendin-4 in PD patients showed that against glutamate-induced apoptosis (Perry et al., exenatide was well tolerated, and the treated group 2003). Furthermore, Val8GLP-1 blocked synaptic deg- exhibited clinically relevant improvements across mo- radation and rescued synaptic plasticity in the hippo- tor and cognitive measures compared with the control campus (Gengler et al., 2012). The neuroprotection effect group (Aviles-Olmos et al., 2013). of GLP-1 was further displayed in studies in which In a study to investigate glucoregulatory effects of treatment with liraglutide, lixisenatide, or geniposide exendins, a research group in Shanghai serendipitously in the transgenic amyloid precursor peptide/presenilin 1 discovered that GLP-1 produced a potent antinocicep- mice produced improvements in synaptic plasticity and tion in the formalin test (Gong et al., 2011). They further reduced neuronal damage, tau hyperphosphorylation, examined the inhibitory role of the spinal GLP-1R microglial activation, plaque, and oligomer formation in signaling pathway in pain hypersensitivity states and the CNS (Perry et al., 2002; McClean et al., 2011; Lv its mechanism of action. Specific expression of GLP-1R et al., 2014; McClean and Holscher, 2014). These GLP-1 on the spinal dorsal horn microglia was found, and the analogs also attenuated memory deficits, restored im- efficacy as well as potency of GLP-1, exenatide, and paired signaling within the Akt/GSK-3b pathway, and geniposide on antinociception in a variety of animal reversed elevated levels of reactive oxygen species in models of pain hypersensitivity versus acute nocicep- amyloid precursor peptide/presenilin 1 mice. The recent tive responses were evaluated (Gong et al., 2014b). The reported effect of liraglutide on an early-stage sporadic involvement of GLP-1 in antinociception was also veri- AD model supports the notion that liraglutide-induced fied by a nonpeptidic GLP-1R agonist (WB4-24) indicat- GLP-1R activity might be a viable target in AD therapy ing that the effect is mediated by GLP-1R (Gong et al., (Hansen et al., 2015). 2014a; Fan et al., 2015). PD is typified by a loss of dopaminergic neurons and 2. Oncological Association. As previously described cellular degeneration in the striatum (Le et al., 2009). above, GLP-1–based therapy is a long-term approach to Epidemiologic data suggest an association between T2DM the control of metabolic disorders exemplified by T2DM, and some neurodegenerative disorders, such as PD whereby chronic adverse events become a major con- (Pressley et al., 2003). In patients with PD, it was found cern, including an increased risk of cancer (Esposito that the levels of insulin receptors were markedly re- et al., 2012). Pancreas and thyroid are the main tissues duced in the basal ganglia and the substantia nigra of the concern. (Moroo et al., 1994). Furthermore, increased IRS2 phos- Since the first case report of exenatide-induced pan- phorylation, a marker of insulin resistance, was found in creatitis in 2006 (Denker and Dimarco, 2006), increasing the basal ganglia of the 6-hydroxydopamine lesion numbers of controversial observations were reported rat PD model (Morris et al., 2008). An earlier report associated with the risk of pancreatitis when treating demonstrated that in high-fat-diet–fed rats, insulin re- T2DM patients with GLP-1R agonists or DPP-4 inhibi- sistance was accompanied by attenuated dopamine re- tors. may potentially progress to lease and diminished dopamine clearance in the basal chronic pancreatitis and ultimately develop pancreatic ganglia (Morris et al., 2011). Neuropathological studies cancer (Yachida et al., 2010; Nauck and Friedrich, 2013). in patients with PD also revealed that loss of insulin Safety alerts have been issued by the FDA for exenatide receptor immunoreactivity and mRNA coincides with and in 2008 and 2009, respectively, for pa- loss of tyrosine hydroxylase mRNA (the rate-limiting tients who have a history of pancreatitis or current enzyme in dopamine synthesis) (Takahashi et al., 1996). symptoms suggestive of pancreatitis. In 2013, a work- GLP-1 and exendin-4 have been shown to exert a shop sponsored by the National Institute of Diabetes and neurotrophic effect on 6-hydroxydopamine–treated do- Digestive and Kidney Diseases and the National Cancer pamine neurons (Li et al., 2010). A growing body of Institute on Pancreatitis-Diabetes-Pancreatic Cancer evidence now exists to support the neurotrophic prop- was held in Bethesda, MD (Andersen et al., 2013). Based erties of GLP-1: activating GLP-1R–signaling pathways on current data, neither the GLP-1R agonist liraglutide in neurons leads to proliferation and differentiation of nor the DPP-4 inhibitor sitagliptin established a solid cells from precursors into neurons, thereby drawing a link with the increase of risk for pancreatic ductal striking similarity between cellular responses in pan- adenocarcinoma in the therapy of patients with T2DM. creatic b cells and neurons (During et al., 2003). Indeed, Ongoing surveillance will be important in future outcome 1002 de Graaf et al. and epidemiologic studies that will be performed as the of the FDA Adverse Event Reporting System database indications for use of these drugs expand; however, suggests that for several malignancies, excluding pan- existing studies have been reassuring to date. creatic and thyroid , exenatide and sitagliptin The effects of GLP-1R activation by liraglutide on apparently significantly reduced the odds ratios of some human pancreatic cancer cells, MIA PaCa-2 and PANC- special cancers, such as lung cancer, prostate cancer, 1, were studied recently (Zhao et al., 2014). Liraglutide lymphoma/multiple myeloma for exenatide, and colon dose- and time-dependently increased GLP-1R expres- cancer and prostate cancer for sitagliptin (Vigneri et al., sion, arrested cell cycles in the S phase, and induced 2009; Koehler et al., 2011; Nauck and Friedrich, 2013). apoptosis through stimulation of cAMP production and However, the data need to be interpreted with caution inhibition of Akt and ERK1/2 pathways. Pancreatic because the results are based on relatively small numbers tumor growth was also attenuated by liraglutide in a of tumors and most likely are subject to reporting bias. mouse xenograft model in vivo. In patients with pan- creatic cancer, 43.3% were GLP-1R–negative (13 of 30) E. GLP-1R Antagonists and, in GLP-1R–positive tissues, tumor size was in- Under normal physiologic conditions, GLP-1 is se- versely correlated with GLP-1R expression. This is the creted directly by the L cells of the gastrointestinal tract only report of the cytoreductive effect of GLP-1R and, as described in this review, acts predominantly to signaling activated by liraglutide on pancreatic cancer enhance insulin secretion, decrease glucagon secretion, cells (Zhao et al., 2014). and inhibit gastric motility (II. Glucagon-Like Peptide-1). is a relatively rare disease, although Based on these data, the main therapeutic strategy tar- its incidence has increased at an alarming rate in both geting GLP-1R has been development of agonists for the men and women in the United States (Aschebrook- treatment of hyperglycemia. Kilfoy et al., 2011; Udelsman and Zhang, 2014). C cells It is established that the insulinotropic action of constitute a minor fraction of the thyroid mass (0.1%) GLP-1 is highly glucose-dependent such that excessive and are the only cells that produce (Huang GLP-1 secretion or sensitivity will not lead to hypo- et al., 2006). GLP-1R agonists are capable of inducing glycemia. However, clinical studies demonstrate that increased calcitonin and C cell hyper- administration of GLP-1 in the presence of a nonglucose- plasia in the thyroid of wild-type, but not the GLP-1R dependent insulin secretagogue (e.g., a sulphonylurea 2/2 knockout (Glp-1r ) mice (Yamada et al., 2008; that acts on the KATP channel) or even directly infusing Madsen et al., 2012). Long-term exposure to GLP-1R supraphysiological levels of GLP-1 into normal subjects agonists resulted in C cell proliferation and the forma- is associated with an increased risk of hypoglycemia tion of C cell adenomas and (medullary thyroid) carci- (Toft-Nielsen et al., 1999; Buse et al., 2004). These data nomas in rodents (Elashoff et al., 2011; Bulchandani suggest that under rare conditions in which either the et al., 2012; Rosol, 2013). Data derived from the FDA secretion of or sensitivity to GLP-1 is significantly en- Adverse Event Reporting System database also indicate hanced, patients may display an increased incidence of a significantly elevated risk for thyroid cancer (histol- insulin secretion and concomitant hypoglycemia. Fur- ogy not specified) with exenatide, but not sitagliptin thermore, these conditions would be predicted to be treatment (Nauck and Friedrich, 2013). Several cases of responsive to treatment with a GLP-1R antagonist. thyroid cancer were reported during the liraglutide GLP-1R has a limited expression profile, and its ago- clinical development program (Neumiller et al., 2010; nists exhibit an excellent safety record, as indicated by Elashoff et al., 2011). Thus, a Black Box warning the successful treatment of patients with T2DM. Fur- regarding the risk of thyroid C cell cancer was labeled thermore, mouse GLP-1R knockouts are viable, develop in all approved long-acting GLP-1 analogs (Parks and normally, and demonstrate no overt phenotype (Scrocchi Rosebraugh, 2010; Andersen et al., 2013). However, a et al., 1996). Therefore, it is perhaps unsurprising that meta-analysis of serious adverse events reported with interest in the potential therapeutic opportunity for GLP- GLP-1R agonists indicates that neither liraglutide nor 1R antagonists has emerged only recently, originating exenatide had an association with an increased risk of largely from a small number of reported clinical condi- thyroid cancer (Alves et al., 2012). Such a discrepancy tions that present severe hypoglycemia. These will be between humans and rodents may reside on the different briefly discussed in the following paragraphs. expression level of GLP-1R in the thyroid because C cells Congenital hyperinsulinism (CHI) represents the most are very sparse in primates when compared with rodents frequent cause of severe, persistent hyperinsulinemic (Gallo, 2013; Pyke et al., 2014). Adequately powered hypoglycemia in newborn babies and children, occur- long-term epidemiologic studies will be necessary to ring in approximately 1/25,000 to 1/50,000 births (Lord clarify the association between GLP-1–based therapies et al., 2015). CHI is caused by genetic defects in key and the risk of thyroid cancer (Andersen et al., 2013). responsible for regulating insulin secretion and Besides the pancreas and thyroid, GLP-1R is widely arises as a consequence of excess insulin secretion from expressed in other tissues, such as the stomach, pitui- the pancreatic b cells (Rahman et al., 2015). Insulin tary, heart, lung, kidney, and nervous system. Analysis directly lowers blood sugars, causing hypoglycemia, Glucagon-Like Peptide-1 and Its Receptors 1003 but insulin hypersecretion also reduces the supply of lead to a profound postprandial hyperinsulinemic hy- alternative sources of energy substrates for oxidative poglycemic state that emerges several years after sur- metabolism in the CNS. As these normally act as a gery (Patti et al., 2005; Ashrafian et al., 2011). protective measure against hypoglycemia, adverse Typically, adult patients experience dizziness, weakness, neurodevelopment outcomes affect approximately one- headache, confusion, lethargy, slurred speech, coma, and third of all patients (Avatapalle et al., 2013). The most seizures depending on the severity and duration of the severe forms of CHI accounting for approximately 45% of hypoglycemic episode. In a landmark paper (Marsk et al., cases are due to recessive inactivating mutations in 2010), the incidence of severe hyperinsulinemic hypogly- ABCC8 and KCJN11 that encode the two components cemia was highlighted in approximately 0.2–0.5% of 5040 of the b cell ATP-sensitive K+ channel (SUR1 and Kir6.2, patients undergoing gastric bypass surgery in Sweden respectively). The current treatment paradigm princi- between 1986 and 2006. Discussion with clinical trust pally involves agonists of KATP channels (diazoxide) and representatives of United Kingdom National Health Ser- somatostatin receptors. However, as the pathology of vice confirmed this as an increasingly recognized unmet CHI mainly involves genetic defects in KATP channels, need, with the figure requiring pharmacological therapy these patients (.50%) will therefore be refractory to after bypass surgery in the United Kingdom likely to be diazoxide treatment that acts via this receptor. Both closer to 1%, in which by the end of 2012 a total of 18,577 targets are expressed on b cells, but also many other cell procedures had been performed under the National types; and this widespread expression profile contributes Health Service. In the United States, the American to multiple off-target complications of these drugs. Phar- Society for Metabolic and Bariatric Surgery reported the macologically unresponsive CHI requires surgical in- number of procedures increased from about 16,000 in the tervention that may be a limited pancreatectomy/ early 1990s to more than 103,000 in 2003, with the total lesionectomy, or in some cases children will require a number of surgeries performed by the end of 2005 exceed- near-total pancreatectomy (95–98%). Despite surgery, ing 590,000. Based on such evidence, it is likely that this patients with the diffuse form of the disease often require condition represents an area of considerable future com- further surgical episodes, and a significant majority of mercial value that is predicted to increase in market size as them will develop iatrogenic and early-onset diabetes. the awareness and related diagnosis continue to expand. The initial link between CHI and GLP-1 was first A number of alternative hypotheses have been sug- reported in rodent models of hyperinsulinism in which gested to explain postprandial hyperinsulinemic hypogly- the KATP channel subunit ABCC8 was knocked out (De cemia, as follows: 1) an exaggerated form of dumping Leon et al., 2008). In this study, treatment with the syndrome; 2) the result of failure of the pancreas to revert GLP-1R antagonist exendin9–39 decreased cAMP levels, after weight loss; and 3) an outcome from altered incretin insulin secretion, and glucose disposal. Interestingly, secretion. Roux-en-Y gastric bypass surgery has been theseeffectsweregeneratedinthefastingstate(i.e., demonstrated to significantly increase the levels of GLP-1 when glucose levels, and hence GLP-1 levels, should secretion. Although the mechanism behind the postsur- have been lowest) and in isolated islets to observe gery hyperinsulinemic hypoglycemia syndrome remains impact on both the basal and stimulated insulin to be confirmed, an emerging number of affected patients secretion. Recently, these findings have been extended present with higher insulin and GLP-1 responses (Service to testing exendin9–39 in adult human subjects with CHI et al., 2005; Salehi et al., 2011). Furthermore, the effects of owing to inactivating mutations in the KATP channel. exogenously administered GLP-1 on gastrointestinal mo- Acute infusion of exendin9–39 significantly increased tility and secretion can be blocked by exendin9–39 (Schirra mean basal glucose levels and glucose area-under-the- et al., 2006). Very recently, Salehi et al. (2014) have curve, and markedly lowered the insulin:glucose ratios reported that this severe postprandial hypoglycemia in (Calabria et al., 2012). Currently, it is unclear in this gastric bypass patients can be corrected by infusion of setting whether the levels of GLP-1 are increased, or exendin9–39, consistent with a fundamental role for GLP-1 whether the responsiveness to exendin9–39 reflects the and its receptor in this mechanism. inverse agonist property of this peptide in controlling Currently, no small-molecule GLP-1R antagonist is excessive insulin secretion by b cells. Recent published available that can be used to further understand the data from the Dunne laboratory in Manchester support consequences of an overactive GLP-1 system. However, the identification of a specific subpopulation with ele- exendin9–39 is a selective, competitive GLP-1R antago- vated GLP-1 levels that may at least partly explain the nist that blocks GLP-1–mediated insulin secretion in vitro pathophysiological role of GLP-1 in this condition. and in vivo and impairs glucose tolerance in response to Roux-en-Y gastric bypass surgery is being used in- endogenous and exogenous GLP-1 in humans and a vari- creasingly in the treatment of morbidly obese type ety of rodent models (Schirra et al., 1998; Edwards et al., 2 diabetic patients, and in the vast majority of patients 1999). Moreover, a number of groups have reported that this results in a very beneficial outcome with significant exendin9–39 inhibits insulin secretion even in the absence weight loss and resolution of diabetes. However, in a of increased GLP-1 levels, suggesting that exendin9–39 small minority of patients, gastric bypass surgery can may be an inverse agonist of GLP-1R (Serre et al., 1998; 1004 de Graaf et al.

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